Monoclonal antibody against connective tissue growth factor and medicinal uses thereof

ABSTRACT

A human monoclonal antibody useful for the treatment of various diseases caused by human connective tissue growth factor (CTGF) and preventing the onset of the above diseases; medicinal uses thereof; and various monoclonal antibodies having various characteristics against various mammalian CTGFs useful for detecting and assaying CTGFs present in body fluids of mammals suffering from various diseases.

CROSS-REFERENCES

This application is a U.S. National Phase application of PCT ApplicationNo. PCT/JP98/05697 filed Dec. 16, 1998, which is the internationalapplication of Japanese Application No. 9/367699 filed Dec. 25, 1997 andJapanese Application No. 10/356,183 filed Dec. 15, 1998, to whichapplications we claim priority.

TECHNICAL FIELD

The present invention relates to monoclonal antibodies reactive tomammalian connective tissue growth factor (CTGF) or a portion thereof;cells producing the monoclonal antibodies; antibody-immobilizedinsoluble carriers on which the monoclonal antibodies or a portionthereof are immobilized; labeled antibodies obtained by labeling themonoclonal antibodies with labeling agents; kits for detecting,separating, assaying or purifying mammalian CTGF; methods for detecting,separating, assaying or purifying mammalian CTGF; pharmaceuticalcompositions comprising the monoclonal antibodies; transgenic mice towhich the human CTGF gene is introduced; a polypeptide of rat CTGF; aDNA encoding rat CTGF; and antibodies reactive to rat CTGF.

BACKGROUND ART

Injured tissues are regenerated by the following process: removal ofuseless tissue fragments and cell fragments or bacteria and so on byphagocytes such as macrophages that migrate to the injured site;recovery of vessels; and the subsequent tissue renewal. Transforminggrowth factor β(TGF-β) produced by macrophages and neutrophils, whichappear during the process of the tissue regeneration and recovery, hasbeen revealed to serve as the first regulatory factor in theregeneration-recovery process.

TGF-β has multiple functions. The factor is known to regulate theproduction of the extracellular matrix (ECM) from connective tissuecells as well as to induce the proliferation of mesenchymal cells and toinhibit the proliferation of vascular endothelial cells and epithelialcells.

Increased production of platelet-derived growth factor (PDGF) andconnective tissue growth factor (CTGF; also called Hcs24) is found inthe culture supernatant of the above-mentioned mesenchymal cells ofwhich proliferation is induced by a stimulus with TGF-β. Because ofthis, it is presumed that the cell proliferation is not directly butindirectly induced by TGF-β with the help of other regulatory factors.

Human and mouse CTGFs have been identified previously (so far, there isno report on the identification of rat CTGF), and their physicochemicaland biological properties have been analyzed (<human CTGF>: J. CellBiology, vol. 114, No. 6, p.1285-1294, 1991; Int. J. Biochem. CellBiol., Vol. 29, No. 1, p. 153-161, 1997; Circulation, vol. 95, No. 4,p.831-839, 1997; Cell Growth Differ., Vol. 7, No. 4, p. 469-480, 1996;J. Invest. Dermatol., Vol. 106, No. 4, p. 729-733, 1996; J. Invest.Dermatol., Vol. 105, No. 2, p. 280-284, 1995; J. Invest. Dermatol. Vol.105, No. 1, p. 128-132, 1995; WO96/38172; <mouse CTGF (Fisp12)>:Unexamined Published Japanese Patent Application (JP-A) No. Hei5-255397; Cell Growth Differ., vol. 2, No. 5, p. 225-233, 1991; FEBSLetters, Vol. 327, No. 2, p. 125-130, 1993; DNA Cell Biol., Vol. 10, No.4, p. 293-308, 1991).

CTGF is a cysteine-rich secretory glycoprotein with a molecular weightof about 38 kDa. It has been revealed that the biosynthesis andsecretion of the protein are induced by TGF-β. CTGF has similarproperties with PDGF in the light of that: their productions are inducedby TGF-β; they bind to the PDGF receptor and induce the proliferation ofmesenchymal cells; and they are produced by fibroblasts and epithelialcells. However, they exhibit no homology at the amino acid level andthus the two molecules are distinct to each other (The Journal of CellBiology, vol. 114, No. 6, p. 1287-1294, 1991; Molecular Biology of theCell, Vol. 4, p.637-645, 1993).

In recent studies, low molecular weight species of CTGF have been foundin the culture supernatant of human and mouse fibroblast cells as wellas in the secreting fluid derived from the porcine uterus. They arebiologically active but are presumed to be degradation products of 38kDa CTGF molecules, since their molecular weights are about 10-12 kDa(Growth Factors, vol. 15, No. 3, p. 199-213, 1998; J. Biol. Chen., vol.272, No. 32, p. 20275-20282, 1997).

Details of the relationship between physiological functions of CTGF anddiseases have yet to be fully clarified. However, it has been foundthat: CTGF production is induced by TGF-β; the expression level of CTGFmRNA is significantly high in tissues and cells derived from patientsaffected with various diseases (Int. J. Biochem. Cell. Biol., Vol. 29,No. 1, p. 153-161, 1997; Circulation, Vol. 95, No. 4, p. 831-839, 1997;J. Invest. Dermatol, Vol. 106, No. 4, p. 729-733, 1996; J. Invest.Dermatol., Vol. 105, No. 2, p. 128-132, 1995; J. Cell Physiol., Vol.165, No. 3, p. 556-565, 1995; Kidney Int., Vol. 48, No. 2, p. 5001-5009,1995); and CTGF enhances the chemotaxis and proliferation of thevascular endothelial cells (J. Cell. Biol., Vol. 114, No. 6, p.1285-1294, 1991; Exp. Cell Res., Vol. 233, p. 63-77, 1997; Journal ofJapanese Association for Oral Biology, Vol. 38, extra number, p. 463,PD0187, 1996; the 69th meeting of the Japanese Biochemical Society,proceedings, p. 683, 1P0535, 1996). These findings suggest thepossibility that CTGF is associated with the onset and/or advancement ofa variety of diseases.

Identification of the specific diseases awaits further findings andadvancement in research. Nonetheless, CTGF has been presumed to beinvolved in the onset and/or advancement of a wide variety of diseasesincluding, for example, cancers, arteriosclerosis, and skin diseases(for example, psoriasis, scleroderma, atopy, and keloid), kidneydiseases, arthritis (for example, rheumatoid arthritis), variousfibrotic diseases (fibrotic diseases in tissues as observed inarteriosclerosis, cirrhosis, arthritis, scleroderma, keloid, kidneyfibrosis and pulmonary fibrosis, etc.).

To elucidate the association of CTGF with such various diseases, it isgenerally effective to detect and assay CTGF and/or the proteinfragments thereof in the body fluids (serum, etc.) from patients andmammals affected with the diseases; the values determined are comparedwith normal values (obtained from mammals including normal persons,normal mice, normal rats and normal rabbits, etc.).

The detection and assay of secretory proteins such as CTGF are carriedout by immunological assays based on antigen-antibody interaction byusing the antibody (preferably used are monoclonal antibodies) which isreactive to the secretory protein to be detected; specifically,immunoassays such as radioimmunoassay (RIA) and enzyme immunoassay (EIA,ELISA) are widely used as the most convenient and useful methods for thepurpose.

In this context, for the purpose of assaying CTGF, it is necessary todevelop detection and assay methods using such immunoassay systems andalso to prepare monoclonal antibodies against CTGF required for theestablishment of assay methods. There are some reports on thepreparation of antiserum reactive to CTGF (Exp. Cell Res., Vol. 233, p.63-77, 1997; Cell Growth Differ., Vol. 8, No. 1, p. 61-68, 1997; the69th meeting of Japanese Biochemical Society, proceedings, p. 683,1P0534, 1996) but no report on the preparation of functional anti-CTGFmonoclonal antibody which has particularly high affinity for CTGF and/orthe capability of neutralizing the CTGF activity; no immunoassay systemsfor CTGF have so far been provided at all.

Such monoclonal antibodies having the capability of neutralizing theCTGF activity described hereinabove are useful not only as components inan immunoassay system but also as pharmaceutical antibody preparationsfor the treatment and/or prevention of the above-mentioned diseasescaused by CTGF secretion. However, there have not been any report onsuch monoclonal antibodies yet.

DISCLOSURE OF THE INVENTION

Thus, the development of monoclonal antibodies reactive to CTGFs fromvarious mammals such as humans, mice, rats and rabbits, has beendesirably awaited. Such monoclonal antibodies are useful for theunderstanding of biological functions of CTGF associated with the onsetand/or advancement of the above-mentioned various diseases as well asfor the understanding of cause-effect relations between CTGF and thevarious diseases. Such monoclonal antibodies are also usable as activeingredients of pharmaceutical products for treating and preventing thediseases caused by CTGF. In particular, development of monoclonalantibodies having sufficiently high affinities for CTGF, the capabilityof neutralizing the CTGF activity, and/or the sufficient crossreactivityto CTGFs from a variety of mammalian species, is demanded when theantibodies are used as components in immunoassay systems for detectingCTGF to elucidate the functions of CTGF as well as the relationshipbetween CTGF and various diseases.

In addition, it is necessary to develop monoclonal antibodies withreduced antigenicity or without antigenicity as well as with theneutralizing activity described above, when the antibodies are used forthe treatment and/or prevention of various diseases in patients.

In order to fulfill the social needs, the present inventors extensivelystudied the monoclonal antibodies against CTGFs from a variety ofmammals, and using CTGFs from various mammals as immunogens, succeededin preparing various monoclonal antibodies against CTGFs from a varietyof mammals; the antibodies are different in properties such as antigenicspecificity, affinity for the antigens, the neutralizing activity andthe crossreactivity.

The present inventors also succeeded, for the first time in the world,in preparing various human monoclonal antibodies against human CTGF, byimmunizing, with human CTGF as an immunogen, transgenic mice created toproduce human antibodies by using recombinant technology. Furthermore,the present inventors found that intact CTGFs in body fluids (serum,etc.) from a variety of mammals (human, mouse, rat, and rabbit) could behighly sensitively assayed by using various immunoassay systemsconstructed with the various monoclonal antibodies described above. Thusthe present inventions were achieved.

The present invention was also achieved by the findings that the latter,i.e., the human antibodies, has not only the capability of significantlyneutralizing the human CTGF activity, but also therapeutic effects on,for example, fibrotic diseases in tissues (kidney fibrosis, etc.) aswell. The fact that these human antibodies are non-antigenic in humans,dramatically elevates the utility value of antibody as a pharmaceutical,because antigenicity is a major therapeutic problem (side effect) inmedical treatment with antibody pharmaceuticals comprising antibodiesderived from non-human mammals such as mice.

In particular, the present invention provides, for the first time in thefield to which the present invention pertains, monoclonal antibodiesthat are reactive to various mammalian CTGFs and possess various usefulproperties as pharmaceuticals to treat and prevent diseases in patientsand as components in immunoassay systems to detect and assay CTGF inbody fluids from various mammals such as humans, mice, and rats.

In addition to this, the present invention provides methods and kits ofimmunoassay for CTGF using such various monoclonal antibodies againstCTGF for the first time.

The inventive anti-human CTGF monoclonal antibodies are extremely usefulas pharmaceuticals for the treatment and prevention of various diseasescaused by CTGF because the antibodies are nonantigetic in humans.

By using an immunoassay with the monoclonal antibodies of the presentinvention, it is possible to conveniently and highly sensitively detectand assay intact CTGF in the body fluids from healthy and diseasedmammals (humans, mice, rats and rabbits).

Specifically, the present inventions are defined as follows:

(1) a monoclonal antibody or a portion thereof, comprising a property inany of (a) to (g) below:

(a) reactive to human, mouse and rat connective tissue growth factors(CTGFs);

(b) reactive to both human and mouse CTGFs but not reactive to rat CTGF;

(c) reactive to both mouse and rat CTGFs but not reactive to human CTGF;

(d) inhibiting binding of human CTGF to human kidney-derived fibroblastcell line 293-T (ATCC CRL1573), or the binding of mouse CTGF to saidcell line 293-T;

(e) inhibiting binding of human CTGF to any cells of rat kidney-derivedfibroblast cell line NRK-49F (ATCC CRL-1570), human osteosarcoma-derivedcell line MG-63 (ATCC CRL-1427), or human lung-derived fibroblasts;

(f) inhibiting cell proliferation of rat kidney-derived fibroblast cellline NRK-49F (ATCC CRL-1570)induced by a stimulus with human or mouseCTGF; or,

(g) inhibiting an increase of hydroxyproline in the kidney, wherein saidhydroxyproline level tends to be elevated;

(2) the monoclonal antibody or a portion thereof according to (1),comprising a property in any of (a) to (c) below:

(a) obtainable by immunizing a mouse with human CTGF or a portionthereof, and reactive to human, mouse and rat CTGFS;

(b) obtainable by immunizing a hamster with mouse CTGF or a portionthereof, and reactive to human, mouse and rat CTGFS; or,

(c) obtainable by immunizing a rat with mouse CTGF or a portion thereof,and reactive to human, mouse and rat CTGFS;

(3) the monoclonal antibody or a portion thereof according to (1),comprising a property in any of (a) to (c) below:

(a) obtainable by immunizing a mouse with human CTGF or a portionthereof, reactive to human, mouse and rat CTGFs and inhibiting bindingof human CTGF to human kidney-derived fibroblast cell line 293-T (ATCCCRL1573);

(b) obtainable by immunizing a rat with mouse CTGF or a portion thereof,reactive to human, mouse and rat CTGFs and inhibiting binding of mouseCTGF to human kidney-derived fibroblast cell line 293-T (ATCC CRL1573);or,

(c) obtainable by immunizing a hamster with mouse CTGF or a portionthereof, and reactive to human, mouse and rat CTGFs and inhibitingbinding of mouse CTGF to human kidney-derived fibroblast cell line 293-T(ATCC CRL1573);

(4) the monoclonal antibody or a portion thereof according to (1),wherein said monoclonal antibody is produced by a hybridoma identifiedby an international deposit accession No. FERM BP-6208;

(5) the monoclonal antibody or a portion thereof according to (1),wherein said monoclonal antibody comprises a property substantiallyequivalent to that of a monoclonal antibody produced by a hybridomaidentified by an international deposit accession No. FERM BP-6208;

(6) the monoclonal antibody or a portion thereof according to (1),wherein said monoclonal antibody is produced by a hybridoma identifiedby an international deposit accession No. FERM BP-6209;

(7) the monoclonal antibody or a portion thereof according to (1),wherein said monoclonal antibody comprises a property substantiallyequivalent to that of a monoclonal antibody produced by a hybridomaidentified by an international deposit accession No. FERM BP-6209;

(8) a human monoclonal antibody or a portion thereof, reactive to anyhuman, mouse or rat CTGF;

(9) the human monoclonal antibody or a portion thereof according to (8),wherein said human monoclonal antibody is reactive to human CTGF;

(10) a human monoclonal antibody or a portion thereof, reactive to humanCTGF and comprises a property in any of (a) to (d) below:

(a) inhibiting binding of human CTGF to human kidney-derived fibroblastcell line 293-T (ATCC CRL1573);

(b) inhibiting binding of human CTGF to any of rat kidney-derivedfibroblast cell line NRK-49F (ATCC CRL-1570), human osteosarcoma-derivedcell line MG-63 (ATCC CRL-1427), or human lung-derived fibroblasts;

(c) inhibiting the cell proliferation of rat kidney-derived fibroblastcell line NRK-49F (ATCC CRL-1570)induced by a stimulus with human ormouse CTGF; or,

(d) inhibiting an increase of hydroxyproline in kidney, wherein saidhydroxyproline level tends to be elevated;

(11) the human monoclonal antibody or a portion thereof according to anyone of (8) to (10), wherein said human monoclonal antibody is derivedfrom a non-human transgenic mammal which is capable of producing a humanantibody;

(12) the human monoclonal antibody or a portion thereof according to(11), wherein said human monoclonal antibody is obtainable by immunizinga non-human transgenic mammal which is capable of producing a humanantibody, with human CTGF;

(13) the human monoclonal antibody or a portion thereof according to anyone of (8) to (12), wherein said non-human transgenic mammal is atransgenic mouse;

(14) the human monoclonal antibody or a portion thereof according to anyone of (8) to (13), wherein a V-region DNA encoding a heavy chainvariable region of said human monoclonal antibody is derived from a genesegment selected from the group consisting of DP-5, DP-38, DP-65 andDP-75;

(15) the human monoclonal antibody or a portion thereof according to anyone of (8) to (13), wherein a V-region DNA encoding a light chainvariable region of said human monoclonal antibody is derived from a genesegment selected from the group consisting of DPK1, DPK9, DPK12 andDPK24;

(16) the human monoclonal antibody or a portion thereof according to anyone of (8) to (15), wherein a V-region DNA encoding a heavy chainvariable region of said human monoclonal antibody is derived from a genesegment selected from the group consisting of DP-5, DP-38, DP-65 andDP-75, and wherein a V-region DNA encoding a light chain variable regionof said human monoclonal antibody is derived from a gene segmentselected from the group consisting of DPK1, DPK9, DPK12 and DPK24;

(17) the human monoclonal antibody or a portion thereof according to(9), wherein an amino acid sequence of a heavy chain variable region ofsaid human monoclonal antibody comprises an amino acid sequence definedbelow in any of (a) to (j) below:

(a) the amino acid positions 21 to 120 of the amino acid sequence of SEQID NO: 6;

(b) the amino acid positions 21 to 120 of the amino acid sequence of SEQID NO: 6, wherein one or more amino acids are deleted, substituted,inserted or added;

(c) the amino acid positions 21 to 118 of the amino acid sequence of SEQID NO: 8;

(d) the amino acid positions 21 to 118 of the amino acid sequence of SEQID NO: 8, wherein one or more amino acids are deleted, substituted,inserted or added;

(e) the amino acid positions 21 to 116 of the amino acid sequence of SEQID NO: 10;

(f) the amino acid positions 21 to 116 of the amino acid sequence of SEQID NO: 10, wherein one or more amino acids are deleted, substituted,inserted or added;

(g) the amino acid positions 21 to 116 of the amino acid sequence of SEQID NO: 12;

(h) the amino acid positions 21 to 116 of the amino acid sequence of SEQID NO: 12, wherein one or more amino acids are deleted, substituted,inserted or added;

(i) the amino acid positions 21 to 117 of the amino acid sequence of SEQID NO: 14; or,

(j) the amino acid positions 21 to 117 of the amino acid sequence of SEQID NO: 14, wherein one or more amino acids are deleted, substituted,inserted or added;

(18) the human monoclonal antibody or a portion thereof according to(9), wherein an amino acid sequence of a light chain variable region ofsaid human monoclonal antibody comprises an amino acid sequence in anyof (a) to (j) below:

(a) the amino acid positions 21 to 120 of the amino acid sequence of SEQID NO: 16;

(b) the amino acid positions 21 to 120 of the amino acid sequence of SEQID NO: 16, wherein one or more amino acids are deleted, substituted,inserted or added;

(c) the amino acid positions 21 to 121 of the amino acid sequence of SEQID NO: 18;

(d) the amino acid positions 21 to 121 of the amino acid sequence of SEQID NO: 18, wherein one or more amino acids are deleted, substituted,inserted or added;

(e) the amino acid positions 23 to 117 of the amino acid sequence of SEQID NO: 20;

(f) the amino acid positions 23 to 117 of the amino acid sequence of SEQID NO: 20, wherein one or more amino acids are deleted, substituted,inserted or added;

(g) the amino acid positions 17 to 111 of the amino acid sequence of SEQID NO: 22;

(h) the amino acid positions 17 to 111 of the amino acid sequence of SEQID NO: 22, wherein one or more amino acids are deleted, substituted,inserted or added;

(i) the amino acid positions 23 to 118 of the amino acid sequence of SEQID NO: 24; or,

(j) the amino acid positions 23 to 118 of the amino acid sequence of SEQID NO: 24, wherein one or more amino acids are deleted, substituted,inserted or added;

(19) a monoclonal antibody or a portion thereof, reactive to human CTGF,which is produced by a hybridoma identified by an international depositaccession No. FERM BP-6535;

(20) a monoclonal antibody or a portion thereof, reactive to human CTGFand comprises a property substantially equivalent to that of amonoclonal antibody produced by a hybridoma identified by aninternational deposit accession No. FERM BP-6535;

(21)a monoclonal antibody or a portion thereof, reactive to human CTGF,and which is produced by a hybridoma identified by an internationaldeposit accession No. FERM BP-6598;

(22) a monoclonal antibody or a portion thereof, reactive to human CTGFand comprises a property substantially equivalent to that of amonoclonal antibody produced by a hybridoma identified by aninternational deposit accession No. FERM BP-6598;

(23) a monoclonal antibody or a portion thereof, reactive to human CTGF,which is produced by a hybridoma identified by an international depositaccession No. FERM BP-6599;

(24) a monoclonal antibody or a portion thereof, reactive to human CTGFand comprises a property substantially equivalent to that of amonoclonal antibody produced by a hybridoma identified by aninternational deposit accession No. FERM BP-6599;

(25) a monoclonal antibody or a portion thereof, reactive to human CTGF,which is produced by a hybridoma identified by an international depositaccession No. FERM BP-6600;

(26) a monoclonal antibody or a portion thereof, reactive to human CTGFand comprises a property substantially equivalent to that of amonoclonal antibody produced by a hybridoma identified by aninternational deposit accession No. FERM BP-6600;

(27) a monoclonal antibody or a portion thereof, reactive to human CTGF,and which is non-reactive to a antigen-antibody complex of human CTGFand the monoclonal antibody reactive to human CTGF of (17) or (18);

(28) the monoclonal antibody or a portion thereof according to (27),wherein said monoclonal antibody is a human monoclonal antibody;

(29) a monoclonal antibody or a portion thereof, reactive to rat CTGF;

(30) a recombinant chimeric monoclonal antibody, reactive to human CTGF,and of which a variable region is derived from a variable region of themonoclonal antibody according to any one of (2) to (7), (27) or (29) andof which a constant region is derived from a constant region of a humanimmunoglobulin;

(31) a recombinant humanized monoclonal antibody, reactive to humanCTGF, of which a whole or portion of the complementarity-determiningregions of a hyper-variable region is derived fromcomplementarity-determining regions of the monoclonal antibody of anyone of (2) to (7), (27) or (29), of which framework regions of ahyper-variable region are derived from the framework regions of a humanimmunoglobulin and of which a constant region is derived from a constantregion of a human immunoglobulin;

(32) a cell producing the monoclonal antibody according to any one of(1) to (29);

(33) a cell producing the recombinant monoclonal antibody according to(30) or (31);

(34) the cell according to (32), wherein said cell is a hybridomaobtainable by fusing a mammalian myeloma cell with a mammalian B cellwhich is capable of producing the monoclonal antibody;

(35) the cell according to (32) or (33), wherein said cell is agenetically engineered cell transformed by either one or both of theDNAs encoding a heavy chain and light chain of the monoclonal antibody;

(36) the hybridoma according to (34), wherein said hybridoma isidentified by an international deposit accession No. FERM BP-6535;

(37) the hybridoma according to (34), wherein said hybridoma isidentified by an international deposit accession No. FERM BP-6598;

(38) the hybridoma according to (34), wherein said hybridoma isidentified by an international deposit accession No. FERM BP-6599;

(39) the hybridoma according to (34), wherein said hybridoma isidentified by an international deposit accession No. FERM BP-6600;

(40) the hybridoma according to (34), wherein said hybridoma isidentified by an international deposit accession No. FERM BP-6208;

(41) the hybridoma according to (34), wherein said hybridoma isidentified by an international deposit accession No. FERM BP-6209;

(42) an antibody-immobilized insoluble carrier on which the monoclonalantibody according to anyone of (1) to (31) is immobilized;

(43) the antibody-immobilized insoluble carrier according to (42),wherein said insoluble carrier is selected from the group consisting ofplates, test tubes, tubes, beads, balls, filters and membranes;

(44) the antibody-immobilized insoluble carrier according to (42),wherein said insoluble carrier is a filter or membrane, or that used foraffinity column chromatography;

(45) a labeled antibody which is prepared by labeling the monoclonalantibody of any one of (1) to (31) with a labeling agent capable ofproviding a detectable signal by itself or together with othersubstances;

(46) the labeled antibody according to (45), wherein said labeling agentis an enzyme, fluorescent substance, chemiluminescent substance, biotin,avidin, or radioisotope;

(47) a kit for detecting or assaying mammalian CTGF, comprising at leastone monoclonal antibody, an antibody-immobilized insoluble carrier, anda labeled antibody, which is selected from the group consisting of themonoclonal antibody according to any one of (1) to (31), theantibody-immobilized insoluble carrier according to (42) or (43), andthe labeled antibody according to (45) or (46);

(48) the kit for detecting or assaying mammalian CTGF according to (47),comprising the antibody-immobilized insoluble carrier according to (42)or (43) and the labeled antibody according to (45) or (46);

(49) a method for detecting or assaying mammalian CTGF by an immunoassayusing at least one monoclonal antibody, an antibody-immobilizedinsoluble carrier, and a labeled antibody, which is selected from thegroup consisting of the monoclonal antibody according to any one of (1)to (31), the antibody-immobilized insoluble carrier according to (42) or(43), and the labeled antibody according to (45) or (46);

(50) the method for detecting or assaying mammalian CTGF by animmunoassay according to (49), comprising at least the following stepsof (a) and (b):

(a) reacting a sample with the antibody-immobilized insoluble carrieraccording to (42) or (43); and,

(b) reacting the labeled antibody according to (45) or (46) with anantigen-antibody complex formed by binding mammalian CTGF in said sampleto the antibody-immobilized insoluble carrier;

(51) the method for detecting or assaying mammalian CTGF by animmunoassay according to (49), comprising at least the following stepsof (a) and (b):

(a) reacting a sample with the labeled antibody according to (45) or(46); and,

(b) reacting the antibody-immobilized insoluble carrier according to(42) or (43) with the antigen-antibody complex formed by binding saidlabeled antibody and mammalian CTGF in said sample;

(52) the method for detecting or assaying mammalian CTGF by animmunoassay according to (49), comprising at least the following step of(a):

(a) reacting a mixture comprising the antibody-immobilized insolublecarrier according to (42) or (43), the labeled antibody according to(45) or (46), and a sample;

(53) the method for detecting or assaying mammalian CTGF by animmunoassay according to (49), comprising at least the following step of(a):

(a) reacting a sample and a mammalian CTGF standard labeled with alabeling agent capable of providing a detectable signal by itself ortogether with other substances, with the antibody-immobilized insolublecarrier according to (42) or (43);

(54) the method for detecting or assaying mammalian CTGFs by animmunoassay according to (49), comprising at least the following stepsof (a) and (b):

(a) reacting the monoclonal antibody according to any one of (1) to (31)with a mixture comprising a sample and a mammalian CTGF standard labeledwith a labeling agent capable of providing a detectable signal by itselfor together with other substances; and,

(b) reacting a mammalian antiserum reactive to said monoclonal antibodywith the antigen-antibody complex formed by binding mammalian CTGF insaid sample or said labeled mammalian CTGF standard and said monoclonalantibody;

(55) the method for detecting or assaying mammalian CTGFs by animmunoassay according to (49), comprising at least the following stepsof any of (a) to (c):

(a) reacting the monoclonal antibody according to any of one s (1) to(31) with a sample;

(b) reacting a mammalian CTGF standard labeled with a labeling agentcapable of providing a detectable signal by itself or together withother substances with a reaction product resulted from the reaction instep (a); and,

(c) reacting a mammalian antiserum reactive to said monoclonal antibodywith the antigen-antibody complex formed by binding mammalian CTGF insaid sample or said labeled mammalian CTGF standard, and said monoclonalantibody;

(56) a kit for separating or purifying mammalian CTGF, comprising theantibody-immobilized insoluble carrier according to (42) or (44);

(57) a method for separating or purifying mammalian CTGF, comprisingusing affinity chromatography with the antibody-immobilized insolublecarrier according to (42) or (44);

(58) the purification method for mammalian CTGF according to (57),wherein said affinity chromatography is affinity column chromatography;

(59) a transgenic mouse in which DNA encoding human CTGF is integratedinto an endogenous gene locus;

(60) a rat CTGF comprising an amino acid sequence of, or substantiallyequivalent to the amino acid sequence of SEQ ID NO: 2;

(61) a DNA encoding a rat CTGF comprising the amino acid sequence of SEQID NO: 2;

(62) the DNA according to (61), comprising nucleotide sequence in thepositions of 213 to 1256 of SEQ ID NO: 1;

(63) a pharmaceutical composition comprising the monoclonal antibody ora portion thereof according to any one of (2) to (31) and apharmaceutically acceptable carrier;

(64) a pharmaceutical composition comprising the human monoclonalantibody or a portion thereof according to any one of (9) to (18) or(28) and a pharmaceutically acceptable carrier;

(65) a pharmaceutical composition comprising the human monoclonalantibody or a portion thereof according to any one of (14) to (18) and(28);

(66) the pharmaceutical composition according to any one of (63) to(65), for inhibiting proliferation of cells capable of proliferating bya stimulus with CTGF;

(67) the pharmaceutical composition according to any one of (63) to(65), for treating or preventing a disease accompanied by proliferationof cells capable of proliferating by a stimulus with CTGF;

(68) the pharmaceutical composition according to (66) or (67), whereinsaid proliferation is cell proliferation in a tissue selected from thegroup consisting of brain, neck, lung, heart, liver, pancreas, kidney,stomach, large intestine, small intestine, duodenum, bone marrow,uterus, ovary, testis, prostate gland, skin, mouth, tongue and bloodvessels;

(69) the pharmaceutical composition according to (68), wherein saidtissue is the lung, liver, kidney or skin;

(70) the pharmaceutical composition according to (69), wherein saidtissue is the kidney;

(71) the pharmaceutical composition according to (67), wherein saiddisease is further accompanied by tissue fibrosis;

(72) the pharmaceutical composition according to (71), wherein saidtissue fibrosis is tissue fibrosis in lung, liver, kidney or skin;

(73) the pharmaceutical composition according to (72), wherein saidtissue fibrosis is kidney fibrosis;

(74) a pharmaceutical composition for treating or preventing a kidneydisease, comprising a CTGF inhibitor or an agent for inhibiting CTGFproduction, and a pharmaceutically acceptable carrier;

(75) the pharmaceutical composition according to (74), wherein saidinhibitor is a monoclonal antibody reactive to CTGF;

(76) the pharmaceutical composition according to (74), wherein saidinhibitor is the monoclonal antibody of any one of (9) to (31);

(77) the pharmaceutical composition according to (76), wherein saidinhibitor is the human monoclonal antibody according to any one of (14)to (18) and (28);

(78) the pharmaceutical composition according to any one of (74) to(77), wherein said disease is accompanied by tissue fibrosis;

(79) a pharmaceutical composition for inhibiting proliferation of cellsin kidney which are capable of proliferating by a stimulus with CTGF,comprising a substance having an activity of inhibiting proliferation ofsaid cells and a pharmaceutically acceptable carrier;

(80) the pharmaceutical composition according to (79), wherein saidsubstance is a monoclonal antibody reactive to CTGF;

(81) the pharmaceutical composition according to (79), wherein saidinhibitor is the monoclonal antibody according to any one of (9) to(31);

(82) the pharmaceutical composition according to (81), wherein saidinhibitor is the human monoclonal antibody according to any one of (14)to (18) and (28).

The present inventions are described in detail herein below by definingterminologies used herein.

Herein, “mammals” mean humans, bovine, goats, rabbits, mice, rats,hamsters and guinea pigs; preferred are humans, rabbits, rats, hamstersor mice, and particularly preferred are humans, rats, hamsters or mice.

The terminologies “mammals except human” and “non-human mammals” in thepresent invention have the same meaning, and both indicate all theabove-defined mammals except humans.

“Amino acids” used in the present invention mean any amino acid existingin nature and preferably the following amino acids presented byalphabetical triplets or single letter codes used to represent aminoacids. (Gly/G) glycine, (Ala/A) alanine, (Val/V) valine, (Leu/L)leucine, (Ile/I) isoleucine, (Ser/S) serine, (Thr/T) threonine, (Asp/D)aspartic acid, (Glu/E) glutamic acid, (Asn/N) asparagine, (Gln/Q)glutamine, (Lys/K) lysine, (Arg/R) arginine, (Cys/C) cysteine, (Met/M)methionine, (Phe/F) phenylalanine, (Tyr/Y) tyrosine, (Trp/W)tryptophane, (His/H) histidine, (Pro/P) proline.

The term “connective tissue growth factor (CTGF)” as referred to in thepresent invention means CTGF derived from the above-mentioned mammals,and includes, for example, human and mouse CTGFs having theabove-described structure and function as reported in previous reports(for example: The Journal of Cell Biology Vol. 114, No. 6, p. 1287-1294,1991; Molecular Biology of the Cell, Vol. 4, p. 637-645, 1993; Biochem.Biophys. Res. Comm. Vol.234, p.206-210, 1997, etc.). As a matter ofcourse, the “connective tissue growth factor” also includes rat CTGFthat is included within the scope of the present invention.

Moreover, “connective tissue growth factor” as referred to in thepresent invention includes not only the CTGF (for example, human CTGF)with a molecular weight of about 38 kDa as documented in reports butalso a low-molecular-weight CTGF protein, with a molecular weightranging from about 10 to about 12 kDa. The low-molecular-weight proteinis assumed to be a degradation product of the full-length CTGF (forexample, human CTGF) with a molecular weight of about 38 kDa (GrowthFactors, Vol. 15, No. 3, p. 199-213, 1998; J. Biol. Chem., Vol. 272, No.32, p. 20275-20282, 1997). Although the structure of thislow-molecular-weight CTGF remains to be clarified, there is apossibility that, in the case of human CTGF, the low-molecular-weightCTGF corresponds to a C-terminal protein (molecular weight: about 11,800Da) consisting of 103 amino acid residues resulted from the cleavage ofthe full-length human CTGF consisting of 349 amino acids between leucineat amino acid position 246 (Leu246) and glutamic acid at amino acidposition 247 (Glu247) or another C-terminal protein (molecular weight:about 11,671 Da) consisting of 102 amino acid residues resulted from thecleavage of the full-length human CTGF between glutamic acid at aminoacid position 247 (Glu247) and glutamic acid at amino acid position 248(Glu248).

In addition, CTGF as referred to in the present invention includes CTGFshaving substantially the same amino acid sequence as that of the naturalCTGF (in particular, human CTGF) having the native primary structure(amino acid sequence) or a portion thereof, as long as the “monoclonalantibody” of the present invention, which is described hereinafter, isreactive to the natural CTGF or a portion thereof.

Here, “having substantially the same amino acid sequence” means toinclude a protein having an amino acid sequence where multiple aminoacids, preferably 1 to 10 amino acids, particularly preferably 1 to 5amino acids, in the amino acid sequence of the natural CTGF protein, aresubstituted, deleted and/or modified, and a protein having an amino acidsequence where multiple amino acids, preferably 1 to 10 amino acids,particularly preferably 1 to 5 amino acids, are added to the amino acidsequence, as long as the protein has substantially the same biologicalproperties as the natural CTGF protein. Furthermore, a combination oftwo or more of the above alterations including a substitution, deletion,modification and addition is also included.

The CTGF of the present invention can be produced by suitably using amethod known in the technical field, such as recombinant technology,chemical synthesis or cell culture, or by using a modified methodthereof.

The CTGF of the present invention also includes “a portion” of the CTGF.The terminology “a portion of CTGF” here refers to a polypeptidecomprising any arbitrary partial amino acid sequence derived from theabove-defined CTGF (including the above-mentioned low-molecular-weightCTGF of about 10 to 12 kDa). Specifically, the polypeptide includes CTGFpeptide fragments with 5 to 100 amino acid residues (for example, thepeptides in the C-terminus), more specifically, includes CTGF peptidefragments with 5 to 50 amino acid residues, and even more specificallythe peptide fragments with 5 to 30 amino acid residues. Preferably, thepolypeptide has a partial structure of CTGF comprising a domain thatbinds or interacts with the receptor thereof (receptor binding site,etc.) or comprising a domain necessary to the biological function ofCTGF (active site, etc.).

These polypeptides (partial structures or fragments) can be producedaccording to a method known in the technical field, or a modified methodthereof, by using recombinant technology or chemical synthesis. Thepolypeptides can also be produced by appropriately digesting the CTGFisolated by the cell culture method with proteases and such.

“Monoclonal antibody” as referred to in the present invention is amonoclonal antibody reactive to mammalian connective tissue growthfactor (CTGF) or a portion thereof. Specifically, the “monoclonalantibody” is a monoclonal antibody having a property described above inany of the inventions (1) to (31). More specifically, “monoclonalantibody” means the various monoclonal antibodies with a variety ofproperties and industrial utilities described below in the examples andas indicated in the drawings.

As a preferable embodiment, the monoclonal antibody of the presentinvention is exemplified by the following monoclonal antibodiesdescribed in (i) to (iv):

(i) the monoclonal antibody according to (1), wherein the monoclonalantibody comprises a property described in any of (d) to (g);

(ii) the monoclonal antibody according to (2);

(iii) the monoclonal antibody according to any one of (4) to (7);

(iv) the monoclonal antibody according to any one of (9) to (31).

In this embodiment, for the purpose of usage as a pharmaceutical fortreating or preventing various diseases, preferable monoclonal antibodyis a human monoclonal antibody included by the antibodies describedabove in (i) to (iv).

In this embodiment, any of the monoclonal antibodies described above in(i) to (iv) are usable for the detection, assay, separation orpurification of mammalian CTGFs, which is another subject matter of thepresent invention.

As a more preferable embodiment, the monoclonal antibody of the presentinvention is exemplified by the following monoclonal antibodiesdescribed in (v) and (vi):

(v) the monoclonal antibody according to (1), wherein the monoclonalantibody comprises a property described in any of (d) to (g);

(vi) the monoclonal antibody according to any of (4) to (7), (10), and(14) to (28).

In this embodiment, for the purpose of usage as a pharmaceutical fortreating or preventing various diseases, preferable monoclonal antibodyis a human monoclonal antibody included in the antibodies describedabove in (v) and (vi).

Furthermore, in this embodiment, any of the monoclonal antibodiesdescribed above in (v) and (vi) are usable for the detection, assay,separation or purification of mammalian CTGFs which is another subjectmatter of the present invention.

As a particularly preferable embodiment, the monoclonal antibody of thepresent invention is exemplified by the following monoclonal antibodiesdescribed in (vii) and (viii):

(vii) the monoclonal antibodies described above in any of (iv) to (vii);

(viii) the monoclonal antibody according to any of (14) to (26) and(28).

In this embodiment, for the purpose of usage as a pharmaceutical fortreating or preventing various diseases, preferable monoclonal antibodyis the human monoclonal antibody described above in (viii).

In this embodiment, any of the monoclonal antibodies described above in(vii) and (viii)are usable for the detection, assay, separation orpurification of mammalian CTGFs which is another subject matter of thisinvention.

As a more particularly preferable embodiment, the monoclonal antibody ofthe present invention is exemplified by the following monoclonalantibodies described in (ix) to (xiv):

(ix) the monoclonal antibody according to (4) or(6);

(x) the monoclonal antibody according to any of (14) to (16);

(xi) the monoclonal antibody according to (17), wherein the monoclonalantibody comprises a property described in any of (a), (c), (e), (g) and(i);

(xii) the monoclonal antibody according to (18), wherein the monoclonalantibody comprises a property described in any of (a), (c), (e), (g) and(i);

(xiii) the monoclonal antibody according to any of (19), (21) (23) and(25);

(xiv) the monoclonal antibody according to (28).

In this embodiment, for the purpose of usage as a pharmaceutical fortreating or preventing various diseases, preferable monoclonal antibodyis the monoclonal antibody described above in any of (x) to (xiv).

In this embodiment, any of the monoclonal antibodies described above in(ix) to (xiv) are usable for the detection, assay, separation orpurification of mammalian CTGFs, which is another subject matter of thepresent invention. However, the monoclonal antibody described above in(ix) is particularly preferable for the purpose.

The “monoclonal antibody” of the present invention also includes anatural monoclonal antibody prepared by immunizing mammals such as mice,rats, hamsters, guinea pigs or rabbits with the above-defined connectivetissue growth factor (including natural, recombinant, and chemicallysynthesized protein and cell culture supernatant) or a portion thereofas an antigen (immunogen); a chimeric antibody and a humanized antibody(CDR-grafted antibody) produced by recombinant technology; and a humanmonoclonal antibody, for example, obtained by using humanantibody-producing transgenic animals.

The “monoclonal antibody” of the present invention further includes arecombinant monoclonal antibody produced by the “cells producingrecombinant monoclonal antibody” described hereinafter.

The monoclonal antibody includes those having any one of the isotypes ofIgG, IgM, IgA (IgA1 and IgA2), IgD, or IgE. IgG (IgG1, IgG2, IgG3, andIgG4, preferably IgG2 or IgG4) or IgM is preferable. IgG is mostpreferred.

The polyclonal antibody (antisera) or monoclonal antibody of the presentinvention can be produced by known methods. Namely, mammals (includingtransgenic animals generated so as to produce an antibody derived fromanother animal species, such as the human antibody producing transgenicmice described below), preferably, mice, rats, hamsters, guinea pigs,rabbits, cats, dogs, pigs, goats, horses, or bovine, or more preferably,mice, rats, hamsters, guinea pigs, or rabbits are immunized, forexample, with an antigen mentioned above with Freund's adjuvant, ifnecessary. The polyclonal antibody can be obtained from the serumobtained from the animal so immunized. The monoclonal antibodies areproduced as follows. Hybridomas are produced by fusing theantibody-producing cells obtained from the animal so immunized andmyeloma cells incapable of producing autoantibodies. Then the hybridomasare cloned, and clones producing the monoclonal antibodies showing thespecific affinity to the antigen used for immunizing the mammal arescreened.

The antibodies can also be produced using “recombinant monoclonalantibody producing cells” of the present invention described below.

Specifically, the monoclonal antibody can be produced as follows.Immunizations are done by injecting or implanting once or several timesthe CTGF (including natural, recombinant, and synthetic proteins, andcell culture supernatant) or its fragment as mentioned above as animmunogen, if necessary, with Freund's adjuvant, subcutaneously,intramuscularly, intravenously, through the footpad, orintraperitoneally into non-human mammals, such as mice, rats, hamsters,guinea pigs, or rabbits, preferably mice, rats or hamsters (includingtransgenic animals generated so as to produce antibodies derived fromanother animal such as the transgenic mouse producing human antibodydescribed below). Usually, immunizations are performed once to fourtimes every one to fourteen days after the first immunization.Antibody-producing cells are obtained from the mammal so immunized inabout one to five days after the last immunization. The times andinterval of the immunizations can be adequately altered according to theproperties of the immunogen used.

Hybridomas that secrete a monoclonal antibody can be prepared by themethod of Köhler and Milstein (Nature, Vol.256, pp.495-497(1975)) and byits modified method. Namely, hybridomas are prepared by fusingantibody-producing cells contained in a spleen, lymph node, bone marrow,or tonsil obtained from the non-human mammal immunized as mentionedabove, preferably a spleen, with myelomas without autoantibody-producingability, which are derived from, preferably, a mammal such as mice,rats, guinea pigs, hamsters, rabbits, or humans, or more preferably,mice, rats, or humans.

For example, mouse-derived myeloma P3/X63-AG8.653 (653, ATCC No.CRL1580), P3/NSI/1-Ag4-1 (NS-1), P3/X63-Ag8.U1 (P3U1), SP2/0-Ag14(Sp2/0, Sp2), PAI, F0, or BW5147; rat-derived myeloma 210RCY3-Ag.2.3.;or human-derived myeloma U-266AR1, GM1500-6TG-A1-2, UC729-6, CEM-AGR,D1R11, or CEM-T15 can be used as a myeloma used for the cell fusion.

Monoclonal antibody producing cells (e.g., hybridoma) can be screened bycultivating the cells, for example, in microtiter plates and bymeasuring the reactivity of the culture supernatant in the well in whichhybridoma growth is observed, to the immunogen used for the immunizationmentioned above, for example, by an enzyme immunoassay such as RIA andELISA.

The monoclonal antibodies can be produced from hybridomas by cultivatingthe hybridomas in vitro or in vivo such as in the ascites of mice, rats,guinea pigs, hamsters, or rabbits, preferably mice or rats, morepreferably mice and isolating the antibodies from the resulting theculture supernatant or ascites fluid of a mammal.

Furthermore, monoclonal antibodies can be obtained in a large quantityby cloning a gene encoding a monoclonal antibody from a hybridoma or“recombinant monoclonal antibody producing cells” of the presentinvention described below, generating transgenic animals such as bovine,goats, sheep, or pigs in which the gene encoding the monoclonal antibodyis integrated in its endogenous gene using transgenic animal generatingtechnique, and recovering the monoclonal antibody derived from theantibody gene from milk of the transgenic animals (Nikkei Science, No.4,pp.78-84 (1997)).

Cultivating the cells in vitro can be performed depending on theproperty of cells to be cultured, on the object of a test study, and onvarious culture, by using known nutrient media or any nutrient mediaderived from known basal media for growing, maintaining, and storing thehybridomas to produce monoclonal antibodies in the culture supernatant.

Examples of basal media are low calcium concentration media such asHam'F12 medium, MCDB153 medium, or low calcium concentration MEM medium,and high calcium concentration media such as MCDB104 medium, MEM medium,D-MEM medium, RPMI1640 medium, ASF104 medium, or RD medium. The basalmedia can contain, for example, sera, hormones, cytokines, and/orvarious inorganic or organic substances depending on the objective.

Monoclonal antibodies can be isolated and purified from the culturesupernatant or ascites mentioned above by saturated ammonium sulfateprecipitation, euglobulin precipitation method, caproic acid method,caprylic acid method, ion exchange chromatography (DEAE or DE52),affinity chromatography using anti-immunoglobulin column or protein Acolumn.

The monoclonal antibody of the present invention also includes amonoclonal antibody comprising the heavy chain and/or the light chain inwhich either or both of the chains have deletions, substitutions oradditions of one or several amino acids in the sequences thereof;“several amino acids” as referred to here means multiple amino acidresidues, specifically means one to ten amino acid residues, preferablyone to five amino acid residues.

The partial modification of amino acid sequence (deletion, substitution,insertion, and addition) described above, can be introduced into themonoclonal antibody of the present invention by partially modifying thenucleotide sequence encoding the amino acid sequence. The partialmodification of the nucleotide sequence can be performed by the usualmethod of site-specific mutagenesis (Proc. Natl. Acad. Sci. USA, Vol.81, p. 5662-5666, 1984).

“Human monoclonal antibody” as referred to in this invention is a humanmonoclonal antibody reactive to the above-defined mammalian CTGFs(preferably human CTGF). The human monoclonal antibody is exemplified bythe various human monoclonal antibodies with a variety of propertiesdescribed below in the examples and as indicated in the drawings.

Specifically, the monoclonal antibody is a human immunoglobulin which isencoded by the human immunoglobulin gene segments in the entire regionthereof including the variable region of the heavy chain (H chain), theconstant region of the H chain, the variable region of the light chain(L chain) and the constant region of the L chain. The L chain isexemplified by a human κ chain and a human λ chain.

The human monoclonal antibody of the present invention can be produced,for example, by immunizing, with the above-defined mammalian CTGFs,“non-human transgenic mammals which are capable of producing humanantibodies” such as “transgenic mice which are capable of producinghuman antibodies” which can be produced by previously reported methods.By using the above-mentioned usual methods, it is possible to immunizenon-human mammals, to prepare and screen hybridomas producing theantibodies, and to prepare the human monoclonal antibody in largequantities (Nature Genetics, Vol. 7, p. 13-21, 1994; Nature Genetics,Vol. 15, p. 146-156, 1997; Published Japanese Translation of PCTInternational Publication No. Hei 4-504365; Published JapaneseTranslation of PCT International Publication No. Hei7-509137; NikkeiScience, June edition, p.40-50, 1995; WO94/25585; Nature, Vol. 368, p.856-859, 1994; Published Japanese Translation of PCT InternationalPublication No. Hei 6-500233, etc.).

The human antibody-producing transgenic mice can be produced,specifically, for example, via the following processes; other humanantibody-producing non-human transgenic mammals can be produced in thesame manner.

(1) A process for preparing knockout mice in which endogenousimmunoglobulin heavy chain gene has been functionally inactivated andthe inactivation is done by substituting at least a portion of theendogenous gene locus of the mouse immunoglobulin heavy chain for adrug-resistance gene (the neomycin resistance gene, etc.) throughhomologous recombination;

(2) A process for preparing knockout mice in which endogenous gene ofimmunoglobulin light chain (a κ chain gene in particular) has beenfunctionally inactivated and the inactivation is done by substituting atleast a portion of the endogenous gene locus of the mouse immunoglobulinlight chain for a drug-resistance gene (the neomycin resistance gene,etc.) through homologous recombination;

(3) A process for preparing transgenic mice in which a desired portionof the human immunoglobulin heavy chain gene locus has been integratedinto a mouse chromosome, by using a vector, such as yeast artificialchromosome (YAC) vector, capable of transporting mega base genes;

(4) A process for preparing transgenic mice in which a desired portionof the human immunoglobulin light chain (a κ gene in particular) genelocus has been integrated into a mouse chromosome, by using a vector,such as YAC vector, capable of transporting mega base genes;

(5) A process for preparing transgenic mice in which both the mouseendogenous heavy chain and light chain gene loci have been functionallyinactivated and both desired portions of the human immunoglobulin heavychain and light chain genes loci have been integrated in a chromosome,of which preparation is achieved by crossbreeding, in arbitrary order,the knockout mice and the transgenic mice described above in (1) to (4).

The knockout mice mentioned above can be prepared by substituting anysuitable region in the mouse endogenous immunoglobulin gene locus for aforeign marker gene (neomycin resistance gene, etc.) through homologousrecombination so that the immunoglobulin gene locus can be inactivatedso as not to cause a rearrangement of the gene locus.

For example, the method designated as positive-negative selection (PNS)can be used for the inactivation with homologous recombination (NikkeiScience, May edition, p. 52-62, 1994).

The functional inactivation of the immunoglobulin heavy chain locus canbe achieved, for example, by introducing a lesion into a portion of theJ region or a portion of the C region (the Cμ region, for example). Thefunctional inactivation of the immunoglobulin light chain locus can alsobe achieved, for example, by introducing a lesion into a portion of theJ region, a portion of the C region, or a region extending from the Jregion to the C region.

The transgenic mouse can be prepared according to the method as usuallyused for producing a transgenic animal (for example, see “Newest Manualof Animal Cell Experiment”, LIC press, Chapter 7, pp.361-408, (1990)).Specifically, for example, a transgenic mouse can be produced asfollows. Hypoxanthine-guanine phosphoribosyl transferase (HPRT)-negativeembryonic stem cells (ES cells) obtained from a normal mouse blastocystis fused with a yeast cell containing an YAC vector, in which the geneencoding human immunoglobulin heavy chain locus or light chain locus, orits fragment and a HPRT gene have been inserted, by spheroplast fusionmethod. ES cells in which the foreign gene has been integrated into themouse endogenous gene are screened by the HAT selection method. Then,the ES cells screened are microinjected into a fertilized egg(blastocyst) obtained from another normal mouse (Proc. Natl. Acad. Sci.USA, Vol.77, No.12, pp.7380-7384 (1980); U.S. Pat. No. 4,873,191). Theblastocyst is transplanted into the uterus of another normal mouse asthe foster mother. Then, chimeric transgenic mice are born from thefoster mother mouse. By mating the chimeric transgenic mice with normalmice, heterozygous transgenic mice are obtained. By mating theheterozygous transgenic mice with each other, homozygous transgenic miceare obtained according to Mendel's laws.

The “chimeric monoclonal antibody” of the present invention is amonoclonal antibody prepared by genetic engineering, whose variableregion is non-human mammal (e.g. mice, rats, hamsters, and so forth)immunoglobulin-derived variable region and whose constant region ishuman immunoglobulin-derived constant region and is exemplified bymouse/human chimeric antibody.

The constant region derived from human immunoglobulin has the amino acidsequence inherent in each isotype such as IgG (IgG1, IgG2, IgG3 andIgG4), IgM, IgA, IgD, and IgE. The constant region of the recombinantchimeric monoclonal antibody of the present invention can be that ofhuman immunoglobulin belonging to any isotype. Preferably, it is theconstant region of human IgG.

The chimeric monoclonal antibody of the present invention can beproduced, for example, as follows. Needless to say, the productionmethod is not limited thereto.

For example, mouse/human chimeric monoclonal antibody can be prepared,by referring to Experimental Medicine: SUPPLEMENT, Vol. 1.6, No.10(1988); and Examined Published Japanese Patent Application (JP-B) No.Hei 3-73280. Namely, it can be prepared by ligating C_(H) gene (C geneencoding the constant region of H chain) obtained from the DNA encodinghuman immunoglobulin to the downstream of active V_(H) genes (rearrangedVDJ gene encoding the variable region of H chain) obtained from the DNAencoding mouse monoclonal antibody isolated from the hybridoma producingthe mouse monoclonal antibody, and by ligating the C_(L) gene (C geneencoding the constant region of L chain) obtained from the DNA encodinghuman immunoglobulin to the downstream of active V_(L) genes (rearrangedVJ gene encoding the variable region of L chain) obtained from the DNAencoding the mouse monoclonal antibody isolated from the hybridoma, andoperably inserting those into the same or different vectors in anexpressible manner, followed by transformation of host cells with theexpression vector, and cultivation of the transformants.

Specifically, DNAs are first extracted from mouse monoclonalantibody-producing hybridoma by the usual method, digested withappropriate restriction enzymes (for example, EcoRI and HindIII),electrophoresed (using, for example, 0.7% agarose gel), and analyzed bySouthern blotting. After the electrophoresed gel is stained, forexample, with ethidium bromide and photographed, the gel is given markerpositions, washed twice with water, and soaked in 0.25M HCl for 15minutes. Then, the gel is soaked in 0.4 N NaOH solution for 10 minuteswith gentle stirring. The DNAs are transferred to a filter for 4 hoursfollowing the usual method. The filter is recovered and washed twicewith 2×SSC. After the filter is sufficiently dried, it is baked at 75°C. for 3 hours, treated with 0.1×SSC/0.1% SDS at 65° C. for 30 minutes,and then soaked in 3×SSC/0.1% SDS. The filter obtained is treated withprehybridization solution in a plastic bag at 65° C. for 3 to 4 hours.

Next, ³²P-labeled probe DNA and hybridization solution are added to thebag and reacted at 65° C. about 12 hours. After hybridization, thefilter is washed under an appropriate salt concentration, reactiontemperature, and time (for example, 2×SSC-0.1% SDS, room temperature, 10minutes). The filter is put into a plastic bag with a little 2×SSC, andsubjected to autoradiography after the bag is sealed. Rearranged VDJgene and VJ gene encoding H chain and L chain of mouse monoclonalantibody respectively are identified by Southern blotting mentionedabove. The region comprising the identified DNA fragment is fractionatedby sucrose density gradient centrifugation and inserted into a phagevector (for example, Charon 4A, Charon 28, λEMBL3, λEMBL4, etc.). E.coli (for example, LE392, NM539, etc.) are transformed with the phagevectorto generate a genomic library. The genomic library is screened byplaque hybridization such as the Benton-Davis method (Science, Vol.196,pp.180-182 (1977)) using appropriate probes (H chain J gene, L chain (κ)J gene, etc.) to obtain positive clones comprising rearranged VDJ geneor VJ gene respectively. By making the restriction map and determiningthe nucleotide sequence of the clones obtained, it is confirmed thatgenes comprising the desired, rearranged V_(H) (VDJ) gene or V_(L) (VJ)gene have been obtained. Separately, human C_(H) gene and human C_(L)gene used for chimerization are isolated. For example, when a chimericantibody with human IgG1 is produced, Cγ₁, gene is isolated as a C_(H)gene, and Cκ gene is also isolated as a C_(L) gene, are isolated. Thesegenes can be isolated from human genomic library with mouse Cγ₁ gene andmouse Cκ gene, corresponding to human Cγ₁ gene and human Cκ gene,respectively, as probes, taking advantage of the high homology betweenthe nucleotide sequences of mouse immunoglobulin gene and that of humanimmunoglobulin gene.

Specifically, DNA fragments comprising human Cκ gene and an enhancerregion are isolated from human λ Charon 4A HaeIII-AluI genomic library(Cell, Vol.15, pp.1157-1174 (1978)), for example, using a 3 kbHindIII-BamHI fragment from clone Ig146 (Proc. Natl. Acad. Sci. USA,Vol.75, pp.4709-4713 (1978)) and a 6.8 kb EcoRI fragment from cloneMEP10 (Proc. Natl. Acad. Sci. USA, Vol.78, pp.474-478 (1981)) as probes.In addition, for example, after human fetal hepatocyte DNA is digestedwith HindIII and fractioned by agarose gel electrophoresis, a 5.9 kbfragment is inserted into λ788 and then human Cγ₁ gene is isolated withthe probes mentioned above.

Using mouse V_(H) gene, mouse V_(L) gene, human C_(H) gene, and humanC_(L) gene so obtained, and taking promoter region and enhancer regioninto consideration, human C_(H) gene is inserted downstream of mouseV_(H) gene and human C_(L) gene is inserted downstream of mouse V_(L)gene in an expression vector such as pSV2gpt or pSV2neo with appropriaterestriction enzymes and DNA ligase following the usual method. In thiscase, chimeric genes of mouse V_(H) gene/human C_(H) gene and mouseV_(L) gene/human C_(L) gene can be respectively inserted into a same ordifferent expression vector.

Chimeric gene-inserted expression vector(s) thus prepared are introducedinto myelomas (e.g., P3×63.Ag8.653 cells or SP210 cells) that do notproduce antibodies by the protoplast fusion method, DEAE-dextran method,calcium phosphate method, or electroporation method. The transformantsare screened by cultivating in a medium containing a drug correspondingto the drug resistance gene inserted into the expression vector and,then, cells producing desired chimeric monoclonal antibodies areobtained.

Desired chimeric monoclonal antibodies are obtained from the culturesupernatant of antibody-producing cells thus screened.

The “humanized monolonal antibody (CDR-grafted antibody)” of the presentinvention is a monoclonal antibody prepared by genetic engineering andspecifically means a humanized monoclonal antibody wherein a portion orthe whole of the complementarity determining regions of thehyper-variable region are derived from the those of the hyper-variableregion from non-human mammal (mouse, rat, hamster, etc.) monoclonalantibody, the framework regions of the variable region are derived fromthose of the variable region from human immunoglobulin, and the constantregion is derived from that from human-immunoglobulin.

The complementarity determining regions of the hyper-variable regionexists in the hyper-variable region in the variable region of anantibody and means three regions which directly binds, in acomplementary manner, to an antigen (complementarity-determiningresidues, CDR1, CDR2, and CDR3). The framework regions of the variableregion mean four comparatively conserved regions intervening upstream,downstream or between the three complementarity-determiningregions(frame work region, FR1, FR2, FR3, and FR4).

In other words, a humanized monoclonal antibody means that in which thewhole region except a portion, or the whole region, of thecomplementarity determining regions of the hyper-variable region of anonhuman mammal-derived monoclonal antibody have been replaced withtheir corresponding regions derived from human immunoglobulin.

The constant region derived from human immunoglobulin has the amino acidsequence inherent in each isotype such as IgG (IgG1, IgG2, IgG3, IgG4),IgM, IgA, IgD, and IgE. The constant region of a humanized monoclonalantibody in the present invention can be that from human immunoglobulinbelonging to any isotype. Preferably, it is the constant region of humanIgG. The framework regions of the constant region derived from humanimmunoglobulin are not particularly limited.

The humanized monoclonal antibody of the present invention can beproduced, for example, as follows. Needless to say, the productionmethod is not limited thereto.

For example, a recombinant humanized monoclonal antibody derived frommouse monoclonal antibody can be prepared by genetic engineering,referring to Published Japanese Translations of PCT InternationalPublication No. Hei 4-506458 and Unexamined Published Japanese PatentApplication (JP-A) No. Sho 62-296890. Namely, at least one mouse H chainCDR gene and at least one mouse L chain CDR gene corresponding to themouse H chain CDR gene are isolated from hybridomas producing mousemonoclonal antibody, and human H chain gene encoding the whole regionexcept human H chain CDR corresponding to mouse H chain CDR mentionedabove and human L chain gene encoding the whole region except human Lchain CDR corresponding to mouse L chain CDR mentioned above areisolated from human immunoglobulin genes.

The mouse H chain CDR gene(s) and the human H chain gene(s) so isolatedare inserted, in an expressible manner, into an appropriate vector sothat they can be expressed. Similarly, the mouse L chain CDR gene(s) andthe human L chain gene(s) are inserted, in an expressible manner, intoanother appropriate vector so that they can be expressed. Alternatively,the mouse H chain CDR gene(s)/human H chain gene(s) and mouse L chainCDR gene(s)/human L chain gene(s) can be inserted, in an expressiblemanner, into the same expression vector so that they can be expressed.Host cells are transformed with the expression vector thus prepared toobtain transformants producing humanized monoclonal antibody. Bycultivating the transformants, desired humanized monoclonal antibody isobtained from culture supernatant.

The “monoclonal antibody” of the invention includes “a portion” of themonoclonal antibody as well. The “portion of an antibody” used in thepresent invention means a partial region of the antibody, preferablymonoclonal antibody of the present invention as mentioned above, andspecifically, means F(ab′)₂, Fab′, Fab, Fv (variable fragment ofantibody), sFv, dsFv (disulfide stabilized Fv), or dAb (single domainantibody) (Exp. opin. Ther. Patents, Vol.6, No.5, pp.441-456 (1996)).

“F(ab′)₂” and “Fab′” can be produced by treating immunoglobulin(monoclonal antibody) with a protease such as pepsin and papain, andmeans an antibody fragment generated by digesting immunoglobulin nearthe disulfide bonds existing between the hinge regions in each of thetwo H chains. For example, papain cleaves IgG upstream of the disulfidebonds existing between the hinge regions in each of the two H chains togenerate two homologous antibody fragments in which an L chain composedof V_(L) (L chain variable region) and C_(L) (L chain constant region),and an H chain fragment composed of V_(H) (H chain variable region) andC_(H)γ1 (γ1 region in the constant region of H chain) are connected attheir C terminal regions through a disulfide bond. Each of these twohomologous antibody fragments is called Fab′. Pepsin also cleaves IgGdownstream of the disulfide bonds existing between the hinge regions ineach of the two H chains to generate an antibody fragment slightlylarger than the fragment in which the two above-mentioned Fab′ areconnected at the hinge region. This antibody fragment is called F(ab′)₂.

The “monoclonal antibody producing cells” or “recombinant monoclonalantibody producing cells” of this invention mean any cells producing theabove-described monoclonal antibody of this invention. Specific examplesinclude the cells described in (1) to (3) below.

(1) monoclonal antibody-producing B cells that are obtainable from theabove-described non-human mammal or human antibody producing transgenicmouse (or other transgenic non-human mammals) that produces a monoclonalantibody reactive with CTGF, which animal can be produced by immunizingthe animal with the above-defined mammalian CTGF (preferably human CTGF)or a portion thereof or cells secreting the CTGF, etc.;

(2) the above-described hybridomas prepared by fusing antibody producingB cells obtained described above with myelomas derived from mammals; and

(3) monoclonal antibody producing transformants (recombinant cells)obtained by transforming other cells than the monoclonal antibodyproducing B cells and hybridomas (e.g. Chinese hamster ovarian (CHO)cells, Baby hamster kidney (BHK) cells, etc.) with genes (either theheavy chain-encoding gene or the light chain encoding gene, or both)encoding the monoclonal antibody isolated from the monoclonal antibodyproducing B cells or hybridomas.

The monoclonal antibody producing transformants (recombinant cells) of(3) mean recombinant cells producing a recombinant product of themonoclonal antibody produced by B cells of (1) or hybridomas of (2).These antibody producing transformants can be produced using knownrecombinant technology as used for the above-described chimericmonoclonal antibody and humanized monoclonal antibody.

The term “monoclonal antibody comprising a property substantiallyequivalent to” as referred to in the present invention indicates that,when biological properties of two monoclonal antibodies are comparedwith each other, one monoclonal antibody is not significantly differentfrom the other in at least the following biological properties:

(1) the reactivity to CTGF derived from a particular animal, which isused as an immunogen for immunizing a non-human mammal to prepare themonoclonal antibody;

(2) the reactivity to any CTGF derived from animals other than theparticular animal (namely, crossreactivity);

(3) the properties measured by a variety of experiments described belowin the examples.

The term “mammalian antiserum” as referred to in the present inventionindicates a serum containing antibody reactive to the monoclonalantibody of the present invention or a portion thereof. The antiserumcan be produced, according to the above-described method described inthe production of monoclonal antibody, by immunizing mammals such asmice, rats, guineapigs, rabbits, goats, pigs orbovine, preferably rats,guinea pigs, rabbits or goats, with the above-mentioned monoclonalantibody or a portion thereof as an immunogen.

The term “insoluble carrier” as referred to in the present inventionindicates a supporting material thereon used for immobilizing themonoclonal antibbdy or a portion thereof (antibody fragment) of thepresent invention, or CTGF in samples (for example, body fluids such asplasma, culture supernatant, supernatant fluids obtained bycentrifugation, etc.) by physical adsorption or chemical bonding.

The insoluble carrier is exemplified below in (A) and (B):

(A) plates, containers having internal spaces such as test tubes ortubes, beads (microbeads in particular), balls, filters or membranes,made of water-insoluble materials, for example, glass or plastics suchas polystyrene resin, polycarbonate resin, silicone resin or nylonresin;

(B) insoluble carriers, used for affinity chromatography, such ascellulose carriers, agarose carriers, polyacrylamide carriers, dextrancarriers, polystyrene carriers, polyvinyl alcohol carriers, poly(aminoacid) carriers or porous silica carriers.

The term “antibody-immobilized insoluble carrier” as referred to in thepresent invention indicates the above-defined insoluble carrier on whichthe monoclonal antibody (or a portion of the antibody, namely anantibody fragment) of this invention is immobilized by physicaladsorption or chemical bonding. These insoluble carriers withimmobilized antibodies are usable for the detection, assay, separationor purification of CTGF in samples (for example, body fluids such asserum and plasma; culture supernatant; the supernatant fluids obtainedby centrifugation, etc.).

The insoluble carriers shown above in (A) can be used for the detectionand the assay; from the standpoint of the simplicity of operation andthe simultaneous processing of many samples, in particular, themulti-well microtiter plates, which are made of plastics and have manywells, such as 96-well microtiter plates or 48-well microtiter plates,are used preferably as the insoluble carrier in the assay for assayingCTGF. The filters or membranes shown above in (A), or the insolublecarriers shown above in (B), are usable for the separation or thepurification.

A “labeling agent capable of providing a detectable signal through thereaction with the labeling agent alone or together with othersubstances” as referred to in this invention means a substance used forconverting the monoclonal antibody or a portion thereof (antibodyfragment) described above, or a CTGF standard into detectable forms; theconversion can be performed by the physical binding or chemical bondingbetween the labeling agent and the monoclonal antibody or a portionthereof, or between the labeling agent and the CTGF standard material.

Specifically, the labeling agent includes enzymes, fluorescentmaterials, chemiluminescent materials, biotin, avidin or radioisotopes,etc., more specifically, enzymes such as peroxidase (for example,horseradish peroxidase), alkaline phosphatase, β-D-galactosidase,glucose oxidase, glucose-6-phosphate dehydrogenase, alcoholdehydrogenase, malate dehydrogenase, penicillinase, catalase,apo-glucose oxidase, urease, luciferase or acetylcholinesterase;fluorescent materials such as fluorescein isothiocyanate,phycobiliprotein, chelating compounds of rare-earth metals, dansylchloride or tetramethylrhodamine isothiocyanate; radioisotopes such as³H, ¹⁴C, ¹²⁵I or ¹³¹I; biotin; avidin; or chemiluminescent materials.

Radioisotopes and fluorescent materials, even when used alone, give adetectable signal. On the other hand, enzymes, chemiluminescentmaterials, biotin, and avidin give no detectable signals, when usedalone. In these cases, one or more substances are needed with thesubstances in order to give a detectable signal. For example, when thesubstance is an enzyme, at least a substrate for the enzyme is necessaryto give a detectable signal. Various types of substrates are selectabledepending on the methods for measuring the enzyme activity (colorimetry,immunofluorescence method, bioluminescence method or chemiluminescencemethod, etc.). For example, hydrogen peroxide is used as a substrate forperoxidase. When biotin is selected, avidin or enzyme-conjugated avidinis used for the reaction with biotin generally but not always. Accordingto needs, various coloring agents are further used for the reactiondepending on the type of the substrate.

The terminologies, “labeled antibody” and “labeled mammalian CTGFstandard” as referred to in the present invention indicate,respectively, monoclonal antibody (or antibody fragment) and CTGFlabeled with the above-mentioned various labeling agents. The labeledantibody and labeled standard can be used to detect, assay, separate orpurify CTGFs in samples (for example, body fluid samples such as serumand plasma; culture supernatants; or the supernatant fluids obtained bycentrifugation, etc.). In the present invention, any of theabove-mentioned labeling agents are usable. However, biotin or enzymessuch as peroxidase are used favorably for the labeling from thestandpoint of the high detection sensitivity or high assay sensitivityand the simplicity of operation.

Being different from a CTGF of an unknown concentration (amount) in asample, the “CTGF standard” is a CTGF isolated previously and thestandard adjustable to any desired concentration thereof to suit thepurpose of each assay. For example, the standard substance can be usedfor the preparation of calibration curves.

The term “immunoassay” as referred to in the present invention means themethod of detecting or assaying the antigens in samples (for example,body fluid samples such as plasma; culture supernatants; or thesupernatant fluids obtained by centrifugation) based on the principle ofantigen-antibody reaction. In the present invention, for theimmunoassay, one or more monoclonal antibodies (or antibody fragment(s))to be used as the antibody in the antigen-antibody reaction are selectedfrom the above-mentioned monoclonal antibodies (or antibody fragment)reactive to the mammalian CTGF of the present invention, theabove-mentioned antibody-immobilized insoluble carrier (or antibodyfragment-immobilized insoluble carrier) and the above-mentioned labeledantibody (or labeled antibody fragment) as well as the antigen is themammalian CTGF, but otherwise previously known immunoassay methods areapplicable in the assay.

Specifically, the immunoassay is exemplified by single antibody solidphase method, two-antibodies liquid phase method, two-antibodies solidphase method, sandwich method, enzyme multiplied immunoassay technique(EMIT method), enzyme channeling immunoassay, enzyme modulator mediatedenzyme immunoassay (EMMIA), enzyme inhibitor immunoassay, immunoenzymometric assay, enzyme enhanced immunoassay or proximal linkageimmunoassay all of which are described in “Enzyme Immunoassay (3rd Ed.,eds., Eiji Ishikawa et al., Igakushoin, 1987); or the one-pot methodwhich is described in JP-B Hei 2-39747.

In this invention, any of these immunoassays can be selectedappropriately to suit each assay purpose. However, from the standpointof the simplicity of operation and/or the economical advantage, andespecially when considering the clinical applicability, the sandwichmethod, the one pot method, the single antibody solid phase method andthe two-antibodies solid phase method are preferably used in thisinvention; more preferable are the sandwich method and the one potmethod. Particularly preferable is the sandwich method using a labeledantibody prepared by labeling the monoclonal antibody of the presentinvention with an enzyme or biotin as well as using anantibody-immobilized insoluble carrier prepared by immobilizing themonoclonal antibody on a multi-well microplate having many wellsthereon, such as a 96-well microplate; another particularly preferablemethod is the one-pot method using a labeled antibody prepared bylabeling the monoclonal antibody of the present invention with an enzymeor biotin as well as using an antibody-immobilized insoluble carrierprepared by immobilizing the monoclonal antibody on beads, such asmicrobeads, or small balls.

A specific example of a particularly preferable embodiment is thesandwich method or the one-pot method using a labeled antibody preparedby labeling the monoclonal antibody “8-86-2,” as indicated in FIG. 1,with an enzyme or biotin, as well as using an antibody-immobilizedinsoluble carrier prepared by immobilizing the monoclonal antibody“8-64-6” or “13-51-2,” as indicated in FIG. 1, on the microplate or themicrobeads.

Human and mouse CTGFs can be detected or quantified in high sensitivityby immunoassays using the monoclonal antibody “8-64-6”-immobilizedinsoluble carrier, in combination with the monoclonal antibody “8-86-2”labeled with an enzyme or biotin. Rat CTGF (first disclosed in thepresent application) and mouse CTGF can be detected or assayed in highsensitivity by the immunoassay using the monoclonal antibody“13-51-2”-immobilized insoluble carrier, in combination with themonoclonal antibody “8-86-2” labeled with an enzyme or biotin.

The sandwich method, the one-pot method, the single antibody solid phasemethod, and the two-antibodies liquid phase method are described indetail herein below.

The sandwich method corresponds to the method described above in (50) ofthe present invention, and specifically, is an immunoassay thatcomprises at least the following steps (a) and (b):

(a) reacting a sample with the antibody-immobilized insoluble carrier ofthe present invention; and

(b) reacting a labeled antibody of the present invention with theantigen-antibody complex formed by the binding between theantibody-immobilized insoluble carrier and mammalian CTGF in the sample.

According to the present invention, a specific example of the method ofassaying human or mouse CTGF is indicated below, in which the“antibody-immobilized insoluble carrier” is an “antibody-immobilizedmicroplate” prepared by immobilizing the monoclonal antibody 8-64-6” asindicated in FIG. 1 on a microplate, and the “labeled antibody” is themonoclonal antibody “8-86-2”, as indicated in FIG. 1, labeled withbiotin or an enzyme such as peroxidase; the method comprises, forexample, the steps described below, but the method is not to beconstrued as being restricted thereto.

Not only mouse CTGF but also rat CTGF (first disclosed in the presentapplication) can be assayed by the same procedures as indicated below,when the “antibody-immobilized insoluble carrier” is an“antibody-immobilized microplate” prepared by immobilizing themonoclonal antibody “13-51-2” as indicated in FIG. 1 on a microplate andthe “labeled antibody” is the monoclonal antibody “8-86-2”, as indicatedin FIG. 1, labeled with biotin or an enzyme such as peroxidase.

(Step 1) preparing an antibody-immobilized microplate by immobilizingthe monoclonal antibody “18-64-6” of the present invention on amicroplate;

(Step 2) reacting a sample such as a human or mouse serum with themonoclonal antibody immobilized on the antibody-immobilized microplateby adding the sample to the microplate;

(Step 3) washing the microplate to remove the unreacted sample from themicroplate;

(Step 4) preparing a labeled antibody by labeling the monoclonalantibody “8-86-2” of the present invention with biotin or an enzyme suchas peroxidase;

(Step 5) reacting the labeled antibody with the antigen-antibody complexformed through the reaction between human or mouse CTGF in the sampleand the monoclonal antibody immobilized on the microplate, by adding thelabeled antibody to the microplate washed in Step 3;

(Step 6) washing out the unreacted labeled antibody from the microplate;

(Step 7) reacting the labeling agent moiety of the labeled antibody witha substrate selected depending on the type of the enzyme used (when thelabeled antibody used in Step 5 is labeled with an enzyme such asperoxidase), avidin or enzyme-conjugated avidin avidin (when the labeledantibody used in Step 5 is labeled with biotin), by adding, if necessarytogether with a coloring agent, the substrate, or avidin orenzyme-conjugated avidin to the microplate;

(Step 8) reacting a substrate for the enzyme selected depending on thetype of the enzyme conjugated with avidin, with the enzyme conjugatedwith avidin, by adding the substrate, when enzyme-conjugated avidin isused in Step 7;

(Step 9) stopping the enzyme reaction and the coloring reaction byadding a reaction stop solution into the reaction mixture of step 7 or8; and

(Step 10) measuring the calorimetric intensity, fluorescence intensityor luminescence intensity.

The one-pot method corresponds to each of the methods described above in(50), (51) and (52) of the present invention.

Specifically, the first is the immunoassay method comprising at leastthe following steps (a) and (b);

(a) reacting a sample with an antibody-immobilized insoluble carrier ofthe present invention; and

(b) reacting a labeled antibody of the present invention with theantigen-antibody complex formed by the binding between theantibody-immobilized insoluble carrier and mammalian CTGF in the sample.

The second is the immunoassay method comprising at least the followingsteps (a) and (b);

(a) reacting a sample with a labeled antibody of the present invention;and

(b) reacting an antibody-immobilized insoluble carrier of the presentinvention with the antigen-antibody complex formed by the bindingbetween the labeled antibody and mammalian CTGF in the sample.

The third is the immunoassay method comprising at least the followingstep (a);

(a) reacting a mixture of an antibody-immobilized insoluble carrier ofthe present invention, a labeled antibody of the present invention and asample.

According to the present invention, a specific example of the method ofassaying human or mouse CTGF is indicated below, in which the“antibody-immobilized insoluble carrier” is “antibody-immobilizedmicrobeads” prepared by immobilizing the monoclonal antibody “8-64-6” asindicated in FIG. 1 on microbeads, and the “labeled antibody” is themonoclonal antibody “8-86-2”, as indicated in FIG. 1, labeled withbiotin or an enzyme such as peroxidase; the method comprises, forexample, the steps described below, but the method is not to beconstrued as being restricted thereto.

Not only mouse CTGF but also rat CTGF (first disclosed in theapplication) can be assayed by the same procedures as indicated below,when the “antibody-immobilized insoluble carrier” is“antibody-immobilized microbeads” prepared by immobilizing themonoclonal antibody “13-51-2” as indicated in FIG. 1 on microbeads andthe “labeled antibody” is the monoclonal antibody “8-86-2”, as indicatedin FIG. 1, labeled with biotin or an enzyme such as peroxidase.

The first method comprises the following steps:

(Step 1) preparing antibody-immobilized microbeads by immobilizing themonoclonal antibody “8-64-6”, of the present invention on microbeads;

(Step 2) reacting a sample such as a human or mouse serum with themonoclonal antibody immobilized on the microbeads by adding the sampleand the antibody-immobilized microbeads together with a buffer solutioninto a container having internal spaces such as a test tube, microplate,or tube;

(Step 3) washing the beads to remove the liquid content from thecontainer;

(Step 4) preparing a labeled antibody by labeling the monoclonalantibody “8-86-2” with biotin or an enzyme such as peroxidase;

(Step 5) reacting the labeled antibody with the antigen-antibody complexformed through the reaction between the human or mouse CTGF in thesample and the monoclonal antibody immobilized on the beads by addingthe labeled antibody into the container containing the beads washed inStep 3;

(Step 6) removing the unreacted labeled antibody by removing the liquidcontent from the container and washing the beads;

(Step 7) reacting the labeling agent moiety of the labeled antibody witha substrate selected depending on the type of the enzyme used (when thelabeled antibody used in Step 5 is labeled with an enzyme such asperoxidase), avidin or the enzyme-conjugated avidin (when the labeledantibody used in Step 5 is labeled with biotin) by adding, if necessarytogether with a coloring agent, the substrate, or avidin orenzyme-conjugated avidin into the container containing the beads washedin Step 6;

(Step 8) reacting a substrate for the enzyme selected depending on thetype of the enzyme conjugated with avidin, with the enzyme conjugatedwith avidin by adding the substrate, when enzyme-conjugated avidin isused in Step 7;

(Step 9) stopping the enzyme reaction and the coloring reaction byadding a reaction stop solution into the reaction mixture of Step 7 orStep 8; and

(Step 10) measuring the calorimetric intensity, fluorescence intensityor luminescence intensity.

The second method comprises the following steps:

(Step 1) preparing a labeled antibody by labeling the monoclonalantibody “8-86-2”, of the present invention with biotin or an enzymesuch as peroxidase;

(Step 2) reacting a sample such as a human or mouse serum with thelabeled antibody by adding the sample and the labeled antibody togetherwith a buffer solution into a container having internal spaces such as atest tube, microplate or tube;

(Step 3) preparing antibody-immobilized microbeads by immobilizing themonoclonal antibody “8-64-6” of the present invention on microbeads;

(Step 4) reacting the monoclonal antibody immobilized on the beads withthe antigen-antibody complex formed through the reaction between thelabeled antibody and human CTGF or mouse CTGF in the sample by addingthe beads into the reaction system in Step 3;

(Step 5) removing the unreacted labeled antibody by removing the liquidcontent from the container and washing the beads;

(Step 6) reacting the labeling agent moiety of the labeled antibody witha substrate selected depending on the type of the enzyme used (when thelabeled antibody used in Step 2 is labeled with an enzyme such asperoxidase), avidin or the enzyme-conjugated avidin (when the labeledantibody used in Step 2 is labeled with biotin) by adding, if necessarytogether with a coloring agent, the substrate, or avidin orenzyme-conjugated avidin into the container containing the beads washedin Step 5;

(Step 7) reacting a substrate selected depending on the type of theenzyme conjugated with avidin, with the enzyme conjugated with avidin byadding the substrate, when enzyme-conjugated avidin is used in Step 6;

(Step 8) stopping the enzyme reaction and the coloring reaction byadding a stop solution to the reaction system in Step 6 or 7; and (Step9) measuring the colorimetric intensity, fluorescence intensity orluminescence intensity.

The third method comprises the following steps:

(Step 1) preparing antibody-immobilized microbeads by immobilizing themonoclonal antibody “8-64-6”, of the present invention on themicrobeads;

(Step 2) preparing a labeled antibody by labeling the monoclonalantibody “8-86-2” of the present invention with biotin or an enzyme suchas peroxidase;

(Step 3) reacting the labeled antibody and a sample such as a human ormouse serum simultaneously with the monoclonal antibody immobilized onmicrobeads by adding the sample and the antibody-immobilized microbeadsprepared in Step 1 and the labeled antibody prepared in Step 2 togetherwith a buffer solution into a container having internal spaces such as atest tube, plate, or tube.

(Step 4) removing the unreacted labeled antibody by removing the liquidcontent from the container and washing the beads;

(Step 5) reacting the labeling agent moiety of the labeled antibody witha substrate selected depending on the type of the enzyme used (when thelabeled antibody used in Step 3 is labeled with an enzyme such asperoxidase), avidin or the enzyme-conjugated avidin (when the labeledantibody used in Step 3 is labeled with biotin) by adding, if necessarytogether with a coloring agent, the substrate, or avidin orenzyme-conjugated avidin into the container containing the beads washedin Step 4;

(Step 6) reacting a substrate selected depending on the type of theenzyme conjugated with avidin, with the enzyme conjugated with avidin byadding the substrate, when enzyme-conjugated avidin is used in Step 5;

(Step 7) stopping the enzyme reaction and the coloring reaction byadding a stop solution to the reaction system in Step 5 or 6; and

(Step 8) measuring the colorimetric intensity, fluorescence intensity orluminescence intensity.

The single antibody solid phase method corresponds to the methoddescribed above in (53) of the present invention, and specifically, theimmunoassay method that comprises at least the following step (a):

(a) reacting a sample and mammalian CTGF standard labeled with alabeling agent capable of providing a detectable signal by itself or byreacting with other substances, with an antibody-immobilized insolublecarrier of the present invention.

A specific example of the method of assaying human or mouse CTGFaccording to the present invention is indicated below, in which the“antibody-immobilized insoluble carrier” is an “antibody-immobilizedmicroplate” prepared by immobilizing the monoclonal antibody “8-64-6” asindicated in FIG. 1 on a microplate and widely used biotin or enzymesuch as peroxidase is used here as a “labeling agent”; the methodcomprises, for example, the steps described below, but the method is notto be construed as being restricted thereto. Not only mouse CTGF butalso rat CTGF (first disclosed in the application) can be assayed by thesame procedures as indicated below, when the “antibody-immobilizedinsoluble carrier” is an “antibody-immobilized microplate” prepared byimmobilizing the monoclonal antibody “13-51-2” as indicated in FIG. 1 ona microplate and widely used biotin or an enzyme such as peroxidase isuse as the “labeling agent.”

(Step 1) preparing an antibody-immobilized microplate by immobilizingthe monoclonal antibody “8-64-6” on a microplate;

(Step 2) preparing a labeled CTGF standard by labeling the standard withbiotin or an enzyme such as peroxidase;

(Step 3) reacting a sample such as a human or mouse serum and thelabeled CTGF standard competitively with the monoclonal antibodyimmobilized on the microplate by adding the sample and the labeledstandard to the microplate;

(Step 4) washing out the unreacted labeled standard from the microplate;

(Step 5) reacting the labeling agent moiety of the labeled standard witha substrate selected from depending on the type of the enzyme used (whenthe labeled standard used in Step 3 is labeled with an enzyme such asperoxidase), avidin or enzyme-conjugated avidin (when the labeledstandard in Step 3 is labeled with biotin) by adding, if necessarytogether with a coloring agent, the substrate, or avidin orenzyme-conjugated avidin to the microplate washed in Step 4;

(Step 6) reacting a substrate selected depending on the type of theenzyme conjugated with avidin, with the enzyme conjugated with avidin byadding the substrate, when enzyme-conjugated avidin is used in Step 5;

(Step 7) stopping the enzyme reaction and the coloring reaction byadding a stop solution to the microplate; and

(Step 8) measuring the colorimetric intensity, fluorescence intensity orluminescence intensity.

The two antibodies solid phase method corresponds to the methodsdescribed above in (54) and (55) of the present invention.

Specifically, the first is the immunoassay method comprising at leastthe following steps (a) and (b):

(a) reacting the monoclonal antibody of the present invention with amixture comprising a sample and a mammalian CTGF standard labeled with alabeling agent capable of providing a detectable signal by itself or byreacting with other substances; and,

(b) reacting a mammalian antiserum reactive to the monoclonal antibodywith the antigen-antibody complex formed through binding of themonoclonal antibody and the mammalian CTGF in the sample or the labeledmammalian CTGF standard.

The second is the immunoassay method comprising at least the followingsteps (a) to (c):

(a) reacting a monoclonal antibody of the present invention with asample;

(b) reacting a mammalian CTGF standard labeled with a labeling agentcapable of providing a detectable signal by itself or by reacting othersubstances, with the reaction mixture in Step (a); and,

(c) reacting a mammalian antiserum reactive to the monoclonal antibodywith the antigen-antibody complex formed through the binding of themonoclonal antibody and the mammalian CTGF in the sample or the labeledmammalian CTGF standard.

A specific example of the method of assaying human or mouse CTGFaccording to the present invention is indicated below, in which the“monoclonal antibody” is the monoclonal antibody “8-64-6” or themonoclonal antibody “8-86-2”, as indicated in FIG. 1 and widely usedbiotin or an enzyme such as peroxidase is used as the “labeling agent”;the method comprises, for example, the steps described below, but themethod is not to be construed as being restricted thereto.

Not only mouse CTGF but also rat CTGF (first disclosed in theapplication) can be assayed by the same procedures as indicated below,when the “monoclonal antibody” is the monoclonal antibody “13-51-2” asindicated in FIG. 1 and widely used biotin or an enzyme such asperoxidase is used as the “labeling agent.”

The first method comprises the following steps:

(Step 1) preparing the labeled CTGF standard by labeling a human ormouse CTGF standard with biotin or an enzyme such as peroxidase;

(Step 2) reacting a sample such as a human or mouse serum and thelabeled CTGF standard prepared in Step 1 competitively with themonoclonal antibody “18-64-6” or “8-86-2” of the present invention byadding a mixture comprising the sample and the labeled CTGF standardinto a container having internal spaces such as a test tube, plate ortube and by subsequently adding thereto the monoclonal antibody;

(Step 3) reacting an antiserum, derived from mammals except mice,reactive to the mouse monoclonal antibody, such as a goat anti-mouseγ-globulin antiserum, with the antigen-antibody complex, formed in Step2, consisting of the monoclonal antibody and the mammalian CTGF in thesample or the labeled mammalian CTGF standard, to give the resultingprecipitated immune-complex;

(Step 4) separating the precipitated complex by the centrifugation ofthe reaction mixture of Step 3;

(Step 5) reacting the labeling agent moiety of the labeled standard witha substrate selected depending on the type of the enzyme used (when thelabeled standard used in Step 2 is labeled with an enzyme such asperoxidase), avidin or the enzyme-conjugated avidin (when the labeledstandard used in Step 2 is labeled with the biotin) by adding, ifnecessary together with a coloring agent, the substrate, or avidin orenzyme-conjugated avidin to the precipitated complex separated in Step4;

(Step 6) reacting a substrate selected depending on the type of theenzyme conjugated with avidin, with the enzyme conjugated with avidin byadding the substrate, when enzyme-conjugated avidin is used in Step 5;

(Step 7) stopping the enzyme reaction and the coloring reaction byadding a stop solution to the reaction system in Step 5 or 6; and,

(Step 8) measuring the colorimetric intensity, fluorescence intensity orluminescence intensity.

The second method comprises the following steps:

(Step 1) preparing a labeled CTGF standard by labeling a human or mouseCTGF standard with biotin or an enzyme such as peroxidase;

(Step 2) reacting a sample such as a human or mouse serum with themonoclonal antibody “8-64-6” or “8-86-2” of the present invention byadding the sample into a container having internal spaces such as a testtube, plate or tube and by subsequently adding thereto the monoclonalantibody;

(Step 3) reacting the labeled CTGF standard prepared in Step 1 with theremaining unreacted monoclonal antibody, by adding the labeled CTGFstandard to the reaction mixture in Step 2;

(Step 4) reacting an antiserum, derived from mammals except mice,reactive to the mouse monoclonal antibody, such as a goat anti-mouseγ-globulin antiserum, with the antigen-antibody complex, formed in Step2, consisting of the monoclonal antibody and the mammalian CTGF in thesample and/or the antigen-antibody complex, formed in Step 3, consistingof the monoclonal antibody and the labeled mammalian CTGF standard, togive the precipitated immune-complex consisting of the antiserum and theantigen-antibody complex;

(Step 5) separating the precipitated complex by the centrifugation ofthe reaction mixture of Step 4;

(Step 6) reacting the labeling agent moiety of the labeled standard witha substrate selected depending on the type of the enzyme used (when thelabeled standard used in Step 3 is labeled with an enzyme such asperoxidase), avidin or the enzyme-conjugated avidin (when the labeledstandard used in Step 3 is labeled with biotin) by adding, if necessarytogether with a coloring agent, the substrate, or avidin orenzyme-conjugated avidin to the precipitated complex separated in Step5;

(Step 7) reacting a substrate selected depending on the type of theenzyme conjugated with avidin, with the enzyme conjugated with avidin byadding the substrate, when enzyme-conjugated avidin is used in Step 6;

(Step 8) stopping the enzyme reaction and the coloring reaction arestopped by adding a stop solution to the reaction system in Step 6 or 7,and;

(Step 9) measuring the colorimetric intensity, fluorescence intensity orluminescence intensity.

The “affinity chromatography” as referred to in the present inventionindicates the method of separating or purifying the materials ofinterest in samples (for example, the body fluid samples such as a serumand plasma; culture supernatants; or the supernatant fluids obtained bycentrifugation, etc.) by utilizing the interaction (affinity) between apair of materials, for example, antigen and antibody, enzyme andsubstrate, or receptor and ligand.

The method of the present invention relates to the method for separatingor purifying the mammalian CTGFs in samples (for example, the body fluidsamples such as a serum and plasma; culture supernatants; or thesupernatant fluids obtained by centrifugation, etc.) by utilizing theantigen-antibody interaction, specifically, the affinity of themonoclonal antibody of the present invention for mammalian CTGFs asantigens; specifically includes,

(1) a method for separating CTGF in samples by immobilizing themonoclonal antibody (or antibody fragment) reactive to mammlian CTGF onthe above-defined insoluble carriers, such as a filter or a membrane andcontacting the sample with the filter or membrane; and

(2) a method for separating or purifying CTGF in the samples, byimmobilizing, in a usual manner (immobilization by physical adsorption,cross-linking to the carrier polymer, trapping in the carrier matrix ornon-covalent bonding, etc.), the inventive monoclonal antibody (or theantibody fragment) reactive to mammalian CTGF on insoluble carriers suchas cellulose carriers, agarose carriers, polyacrylamide carriers,dextran carriers, and polystyrene carriers, polyvinyl alcohol carriers,poly(amino acid) carriers or porous silica carriers; by filling a columnmade of glass, plastics, or stainless, with the insoluble carriers; andby loading and eluting samples (for example, the body fluid samples suchas a serum and plasma; culture supernatants; or the supernatant fluidsobtained by centrifugation, etc.) through the column (for example, thecylindrical column). The method described above in (2) is in particulardesignated as affinity column chromatography.

Any of the insoluble carriers are usable as insoluble carriers foraffinity column chromatography, as long as the monoclonal antibody (orantibody fragment) of the present invention can be immobilized on thecarriers. Such carriers include, for example, commercially availablecarriers such as SEPHAROSE 2B, SEPHAROSE 4B, SEPHAROSE 6B,CNBR-ACTIVATED SEPHAROSE 4B, AH-SEPHAROSE 4B, CH-SEPHAROSE 4B, ACTIVATEDCH-SEPHAROSE 4B, EPOXY-ACTIVATED SEPHAROSE 6B, ACTIVATED THIOL-SEPHAROSE4B, SEPHADEX, CM-SEPHADEX, ECH-SEPHAROSE 4B, EAH-SEPHAROSE 4B,NHS-ACTIVATED SEPHAROSE or THIOPROPYL SEPHAROSE 6B, etc., all of whichare supplied by Pharmacia; BIO-GEL A, CELLEX, CELLEX AE, CELLEX-CM,CELLEX PAB, BIO-GEL P, HYDRAZIDE BIO-GEL P, AMINOETHYL BIO-GEL P,BIO-GEL CM, AFFI-GEL 10, AFFI-GEL 15, AFFI-PREP10, AFFI-GEL HZ,AFFI-PREP HZ, AFFI-GEL 102, CM BIO-GEL A, AFFI-GEL HEPARIN, AFFI-GEL 501OR AFFI-GEL 601, etc., all of which are supplied by Bio-Rad; CHROMAGELA, CHROMAGEL P, ENZAFIX P-HZ, ENZAFIX P-SH OR ENZAFIX P-AB, etc., all ofwhich are supplied by Wako Pure Chemical Industries Ltd.; AE-CELLUROSE,CM-CELLUROSE or PAB CELLUROSE etc., all of which are supplied by Serva.

The term “pharmaceutical composition” as referred to in the presentinvention means a composition useful as a pharmaceutical comprising asan active ingredient the monoclonal antibody of the present invention ora portion thereof, or any of the after-mentioned “CTGF inhibitor”, “CTGFproduction inhibitor” and “substance with the activity to inhibit theCTGF-stimulated proliferation of cells having the capability ofproliferating by CTGF stimulation”, as well as comprising a“pharmaceutically acceptable carrier.”

The “pharmaceutically acceptable carrier” includes an excipient, adiluent, an expander, a disintegrating agent, a stabilizer, apreservative, a buffer, an emulsifier, an aromatic, a colorant, asweetener, a viscosity increasing agent, a flavor, a dissolving agent,or other additives.

Using one or more of such carriers, a pharmaceutical composition can beformulated into tablets, pills, powders, granules, injections,solutions, capsules, troches, elixirs, suspensions, emulsions, orsyrups.

The pharmaceutical composition can be administered orally orparenterally. Other forms for parenteral administration include asolution for external application, suppository for rectaladministration, and pessary, prescribed by the usual method, whichcomprises one or more active ingredient.

The dosage can vary depending on the age, sex, weight, and symptoms of apatient, effect of treatment, administration route, period of treatment,or the kind of active ingredient (protein or antibody mentioned above)contained in the pharmaceutical composition. Usually, the pharmaceuticalcomposition can be administered to an adult in a dose of 10 μg to 1000mg (or 10 μg to 500 mg) per one administration. Depending on variousconditions, the lower dosage may be sufficient in some cases, and ahigher dosage may be necessary in other cases.

In particular, the injection can be produced by dissolving or suspendingthe antibody in a non-toxic, pharmaceutically acceptable carrier such asphysiological saline or commercially available distilled water forinjections by adjusting the concentration to 0.1 μg antibody/ml carrierto 10 mg antibody/ml carrier.

The injection thus produced can be administered to a human patient inneed of treatment in a dose of 1 μg to 100 mg/kg body weight, preferably50 μg to 50 mg/kg body weight, once or more times a day. Examples ofadministration routes are medically appropriate administration routessuch as intravenous injection, subcutaneous injection, intradermalinjection, intramuscular injection, or intraperitoneal injection,preferably intravenous injection.

The injection can also be prepared into a non-aqueous diluent (forexample, propylene glycol, polyethylene glycol, vegetable oil such asolive oil, and alcohols such as ethanol), suspension, or emulsion.

The injection can be sterilized by filtration with abacteria-non-penetratable filter, by mixing bacteriocide, or byirradiation. The injection can be prepared at the time of use. Namely,it is freeze-dried to make a sterile solid composition, and can bedissolved in sterile distilled water for injection or another solventbefore use.

The pharmaceutical composition of the present invention is useful forinhibiting the proliferation of various cells having the capability ofproliferating (for example, various fibroblast cells, various vascularendothelial cells, and others, etc.) in response to the stimulation ofCTGFs from a variety of tissues. Examples of the tissues are, the brain,neck, lung, heart, liver, pancreas, kidney, stomach, large intestine,small intestine, duodenum, bone marrow, uterus, ovary, testis, prostate,skin, mouth, tongue, and blood vessels, and preferably, the lung, liver,kidney or skin.

As described hereinabove, the pharmaceutical composition of the presentinvention can inhibit the proliferation of cells having the capabilityof proliferating in response to the stimulation of CTGF. Therefore, thepharmaceutical composition of the present invention is also useful as apharmaceutical for treating or preventing a variety of diseasesassociated with the cell proliferation in various tissues mentionedabove. Examples of such tissues are the brain, neck, lung, heart, liver,pancreas, kidney, stomach, large intestine, small intestine, duodenum,bone marrow, uterus, ovary, testis, prostate, skin, mouth, tongue, andblood vessels, and preferably, the lung, liver, kidney or skin.

The diseases, to which the pharmaceutical composition of the presentinvention is applicable for the treatment or prevention, are, forexample, fibrotic diseases in various tissues (kidney fibrosis,pulmonary fibrosis, hepatic fibrosis, fibrosis in the skin, etc.),kidney diseases (for example, kidney fibrosis, nephritis, renal failure,etc.), lung diseases (for example, pulmonary fibrosis, pneumonia, etc.),skin diseases (for example, psoriasis, scleroderma, atopy, keloid,etc.), liver diseases (for example, hepatic fibrosis, hepatitis,cirrhosis, etc.), arthritis (for example, rheumatoid arthritis), variouscancers, or arteriosclerosis.

Preferable examples of the diseases are kidney diseases (for example,kidney fibrosis, nephritis, renal failure, etc.), lung diseases (forexample, pulmonary fibrosis, pneumonia, etc.), skin diseases (forexample, psoriasis, scleroderma, atopy, keloid, etc.), liver diseases(for example, hepatic fibrosis, hepatitis, cirrhosis, etc.).

More preferable are kidney diseases (for example, kidney fibrosis,nephritis, renal failure, etc.).

The pharmaceutical composition of the present invention includes thepharmaceutical composition comprising a “CTGF inhibitor,” a “CTGFproduction inhibitor” or a “substance with the activity to inhibit theCTGF-stimulated proliferation of the cells having the capability ofproliferating by CTGF stimulation.”

Each of the “CTGF inhibitor,” the “CTGF production inhibitor,” and the“substance” means a substance having the activity of suppressing orinhibiting the biological function of CTGF, or a substance having theactivity of suppressing or inhibiting the production of CTGF in avariety of cells. Such substances are exemplified by a substance havingany of the following activities:

(1) the activity of suppressing or inhibiting the binding of humankidney-derived fibroblast cells (for example, cell line 293-T (ATCCCRL1573)) to human CTGF, or the binding of the cells to mouse CTGF;

(2) the activity of suppressing or inhibiting the binding of human CTGFwith rat kidney-derived fibroblast cells (for example, cell line NRK-49F(ATCC CRL-1570)), human osteosarcoma cell line MG-63 (ATCC CRL-1427), orhuman lung-derived fibroblast cells;

(3) the activity of suppressing or inhibiting the proliferation of ratkidney-derived fibroblast cells (for example, cell line NRK-49F (ATCCCRL-1570)) in response to the stimulation of human CTGF or mouse CTGF;

(4) the activity of suppressing or inhibiting an increase ofhydroxyproline in the kidney where the synthesis of hydroxyproline leveltends to be increased.

Specifically, the above-mentioned “substance” is exemplified by thefollowing substances:

(a) the above-mentioned monoclonal antibody of the present invention(which is not restricted to the wild-type antibody and the recombinantantibody) or a portion thereof;

(b) antisense DNA;

(c) antisense RNA;

(d) low molecular weight chemical substances (chemically synthesizedcompounds or naturally-occurring substances) other than the substancesindicated in (a) to (c).

The antisense DNA as referred to in the present invention includes a DNAcomprising a partial nucleotide sequence of a DNA encoding the mammalian(preferably human) CTGF protein or a DNA corresponding to the above DNAthat is chemically modified in part, or a DNA comprising a complementarysequence to the partial nucleotide sequence, or a DNA corresponding tothis DNA that is chemically modified in part.

The “partial nucleotide sequence” as referred to here indicates apartial nucleotide sequence comprising an arbitrary number ofnucleotides contained in an arbitrary region of the DNA sequenceencoding the mammalian (preferably human) CTGF protein.

The DNA, hybridizing to a DNA or an RNA encoding the CTGF protein, caninhibit the CTGF production by suppressing transcription of the DNA tomRNA or suppressing the translation of the mRNA into the protein.

The partial nucleotide sequence consists of 5 to 100 consecutivenucleotides, preferably 5 to 70 consecutive nucleotides, more preferably5 to 50 consecutive nucleotides, and still more preferably 5 to 30consecutive nucleotides.

When the DNA is used as an antisense DNA pharmaceutical, the DNAsequence can be modified chemically in part for extending the half-life(stability) of the blood concentration of the DNA administered topatients, for increasing the intracytoplasmic-membrane permeability ofthe DNA, or for increasing the degradation resistance or the absorptionof the orally administered DNA in the digestive organs. The chemicalmodification includes, for example, the modification of the phosphatebonds, the riboses, the nucleotide bases, the sugar moiety, the 3′ endand/or the 5′ end in the structure of the oligonucleotide DNA.

The modification of phosphate bond includes, for example, the conversionof one or more of the bonds to phosphodiester bonds (D-oligo),phosphorothioate bonds, phosphorodithioate bonds (S-oligo), methylphosphonate (MP-oligo), phosphoroamidate bonds, non-phosphate bonds ormethyl phosphonothioate bonds, or combinations thereof. The modificationof the ribose includes, for example, the conversion to 2′-fluororiboseor 2′-O-methylribose. The modification of the nucleotide base includes,for example, the conversion to 5-propynyluracil or 2-aminoadenine.

The antisense RNA as referred to in the present invention includes anRNA comprising a partial nucleotide sequence of an RNA encodingmammalian (preferably human) CTGF protein or an RNA corresponding to theRNA which is chemically modified in part, or an RNA comprising acomplementary sequence to the partial nucleotide sequence or an RNAcorresponding to this RNA which is chemically modified in part.

The “partial nucleotide sequence” as referred to here indicates apartial nucleotide sequence comprising an arbitrary number ofnucleotides contained in an arbitrary region of the RNA sequenceencoding mammalian (preferably human) CTGF protein.

The RNA, hybridizing to a DNA or an RNA encoding the CTGF protein, caninhibit the CTGF production by inhibiting the transcription of the DNAto mRNA or inhibiting the translation of the mRNA into the protein.

The partial nucleotide sequence consists of 5 to 100 consecutivenucleotides, preferably 5 to 70 consecutive nucleotides, more preferably5 to 50 consecutive nucleotides, and still more preferably 5 to 30consecutive nucleotides.

When the RNA is used as an antisense RNA pharmaceutical, the RNAsequence can be modified chemically in part for extending the half-life(stability) of the blood concentration of the RNA administered topatients, for increasing the intracytoplasmic-membrane permeability ofthe RNA, or for increasing the degradation resistance or the absorptionof the orally administered RNA in the digestive organ. The chemicalmodification includes, for example, the modification of the phosphatebonds, the riboses, the nucleotide bases, the sugar moiety, the 3′ endand/or the 5′ end in the structure of the oligonucleotide RNA.

The modification of phosphate bonds includes, for example, theconversion of one or more of the bonds to phosphodiester bonds(D-oligo), phosphorothioate bond, phosphorodithioate bonds (S-oligo),methyl phosphonate (MP-oligo), phosphoroamidate bonds, non-phosphatebonds or methyl phosphonothioate bonds, or combinations thereof. Themodification of the ribose includes, for example, the conversion to2′-fluororibose or 2′-O-methylribose. The modification of the nucleotidebase includes, for example, the conversion to 5-propynyluracil or2-aminoadenine.

The therapeutic effects of the pharmaceutical composition of the presentinvention on various diseases can be examined and evaluated according toa usual method by administering the composition to known animals asdisease models.

For example, evaluation of the therapeutic effect on kidney fibrosis,which is a tissue fibrosis as well as a kidney disease, can be performedby a method using a renal failure model mouse (unilateral ureteralobstruction (UUO) model), in which unilateral ureteral ligationobstructs renal blood filtration in the kidney and results in renalfailure in the mouse. After administration of the inventivepharmaceutical composition to the mouse, the examination is achieved bymeasuring the degree of inhibition of an increase of hydroxyprolineproduction, which is an index of the onset of nephritis and kidneyfibrosis induced by the renal failure. A decrease in the hydroxyprolineconcentration indicates the efficacy of the pharmaceutical compositionfor the treatment of the kidney disease.

By using the model animals described in detail in a previous report(“Preparation of animals as disease models: Testing and experimentalmethods for the development of new drugs” p. 34-46, 1993, TechnologicalInformation Society), the evaluation can be performed for kidneydiseases including, for example, minimal change glomerular disease (forexample, minimal change nephrotic syndrome (MCNS)), focal glomerularsclerosis (FGS), membraneous glomerulonephritis (membranous nephropathy(MN)), IgA nephropathy, mesangial proliferative glomerulonephritis,acute post-streptococcal glomerulonephritis(APSGN, crescentic(extracapillary) glomerulonephritis, interstitial nephritis, or acuterenal failure.

By using the model animals described in detail in the previous report(“Preparation of animals as disease models: Testing and experimentalmethods for the development of new drugs” p. 229-235, 1993,Technological Information Society ), the evaluation can be performed forskin diseases including, for example, injuries, keloid, atopy,dermatitis, scleroderma or psoriasis.

By using the model animals described in detail in the previous report(“Preparation of animals as disease models: Testing and experimentalmethods for the development of new drugs” p. 349-358, 1993,Technological Information Society ), the evaluation can be performed forliver diseases including, for example, hepatitis (for example, viralhepatitis (type A, type B, type C, type E, etc.)), cirrhosis or druginduced hepatic injuries.

For example, the effect on arteriosclerosis and restenosis can beevaluated by using a restenosis model rat, in which thepseudo-restenosis is causeed by percutaneous transluminal coronaryangioplasty(PTCA) with balloon catheter inserted in the aorta.

For example, the effect on tumor growth and metastasis can be confirmedby using mice as cancer metastasis models. The model mice are preparedby transplanting cancer cells into the subcutaneous tissue, caudal vein,spleen, tissues under the renicapsule, peritoneal cavity or cecum walltissue, of commercially available mice including normal mice such asBalb/c mouse, or model mice such as nude mouse and SCID mouse.

The “rat CTGF” of the present invention (specifically, having an aminoacid sequence of, or substantially equivalent to that of SEQ ID NO: 2)and the “DNA encoding rat CTGF” (specifically, comprising the nucleotidesequence spanning from nucleotide position 213 to 1256 of the nucleotidesequence of SEQ ID NO: 1) are defined below, and can be preparedaccording to a usual method as shown below.

Here, the terminology “substantially equivalent” has the meaning definedabove.

The “rat CTGF” of the present invention can be produced by suitablyusing a method known in this technical field, such as chemical synthesisand cell culture as well as the recombinant technique described below,or by using a modified method thereof.

The “DNA” of the present invention indicates the DNA encoding rat CTGF,and includes any nucleotide sequences as long as the nucleotide sequenceencodes rat CTGF of the present invention. Specifically, the DNAincludes any DNAs encoding the polypeptide with the amino acid sequenceof SEQ ID NO:2. In a preferred embodiment, the DNA comprises thenucleotide sequence spanning from nucleotide position 213 to 1256 in thenucleotide sequence of SEQ ID NO: 1 (for example, the DNA having thenucleotide sequence of SEQ ID NO: 1).

The DNA of the present invention includes both cDNA and genomic DNAencoding rat CTGF.

The DNA of the present invention also includes the DNAs consisting ofany codons as long as the codons encodeidentical amino acids.

The DNA of the present invention can be a DNA obtained by any method.For example, the DNA includes complementary DNA (cDNA) prepared frommRNA, DNA prepared from genomic DNA, DNA prepared by chemical synthesis,DNA obtained by PCR amplification with RNA or DNA as a template, and DNAconstructed by appropriately combining these methods.

The DNA encoding the rat CTGF of the present invention can be preparedby the usual methods: cloning cDNA from MRNA encoding rat CTGF,isolating genomic DNA and splicing it, PCR using the cDNA or mRNAsequence as a template, chemical synthesis, and so on.

The DNA encoding the rat CTGF can be prepared by cleaving (digesting)each DNA encoding the rat CTGF as prepared above with an appropriaterestriction enzyme, and linking the obtained DNA fragments, incombination with linker DNA or Tag if necessary, using an appropriateDNA polymerase and such.

cDNA encoding rat CTGF (hereinafter referred to as the desired protein)can be cloned from mRNA by, for example, the method described below.

First, the mRNA encoding the desired protein is prepared from tissues orcells (for example, rat fibroblasts, etc.) expressing and producing thedesired protein. mRNA can be prepared by isolating total RNA by a knownmethod such as quanidine-thiocyanate method (Chirgwin et al.,Biochemistry, Vol.18, p5294, 1979), hot phenol method, or AGPC method,and subjecting it to affinity chromatography using oligo-dT cellulose orpoly-U Sepharose.

Then, with the MRNA obtained as a template, cDNA is synthesized, forexample, by a well-known method using reverse transcriptase, such as themethod of Okayama et al (Mol. Cell. Biol. Vol.2, p.161 (1982); ibid.Vol.3, p.280 (1983)) or the method of Hoffman et al. (Gene Vol.25, p.263(1983)), and converted into double-stranded cDNA. A cDNA library isprepared by transforming E. coli with plasmid vectors, phage vectors, orcosmid vectors having this cDNA or by transfecting E. coli after invitro packaging.

The plasmid vectors used in this invention are not limited as long asthey are replicated and maintained in hosts. Any phage vector that canbe replicated in hosts can also be used. Examples of usually usedcloning vectors are pUC19, λgt10, λgt11, and so on. When the vector isapplied to immunological screening as mentioned below, a vector having apromoter that can express a gene encoding the desired protein in a hostis preferably used.

cDNA can be inserted into a plasmid by, for example, the method ofManiatis et al. (Molecular Cloning, A Laboratory Manual, second edition,Cold Spring Harbor Laboratory, p.1.53, 1989). cDNA can be inserted intoa phage vector by, for example, the method of Hyunh et al. (DNA cloning,a practical approach, Vol.1, p.49 (1985)). These methods can be simplyperformed by using a commercially available cloning kit (for example, aproduct from Takara Shuzo). The recombinant plasmid or phage vector thusobtained is introduced into an appropriate host cell such as aprokaryote (for example, E. coli: HB101, DH5α, Y1090, DH10B, MC1061/P3,etc).

Examples of a method for introducing a plasmid into a host are, calciumchloride method, calcium chloride/rubidium chloride method andelectroporation method, described in Molecular Cloning, A LaboratoryManual (second edition, Cold Spring Harbor Laboratory, p.1.74 (1989)).Phage vectors can be introduced into host cells by, for example, amethod in which the phage DNAs are introduced into grown hosts after invitro packaging. In vitro packaging can be easily performed with acommercially available in vitro packaging kit (for example, a productfrom Stratagene or Amersham).

The cDNA encoding the desired protein can be isolated from the cDNAlibrary so prepared according to the method mentioned above by combininggeneral cDNA screening methods.

For example, a clone comprising the desired cDNA can be screened by aknown colony hybridization method (Crunstein et al. Proc. Natl. Acad.Sci. USA, Vol.72, p.3961 (1975)) or plaque hybridization method(Molecular Cloning, A Laboratory Manual, second edition, Cold SpringHarbor Laboratory, p.2.108 (1989)) using ³²P-labeled chemicallysynthesized oligonucleotides as probes, which correspond to the aminoacid sequence of the desired protein. Alternatively, a clone having aDNA fragment encoding a specific region within the desired protein canbe screened by amplifying the region by PCR with synthetic PCR primers.

When a cDNA library prepared using a cDNA expression vector (forexample, kgt11 phage vector) is used, the desired clone can be screenedby the antigen-antibody reaction using an antibody against the desiredprotein. A screening method using PCR method is preferably used whenmany clones are subjected to screening.

The nucleotide sequence of the DNA thus obtained can be determined byMaxam-Gilbert method (Maxam et al. Proc. Natl. Acad. Sci. USA, Vol.74,p.560 (1977)) or the dideoxynucleotide synthetic chain terminationmethod using phage M13 (Sanger et al. Proc. Natl. Acad. Sci. USA,Vol.74, pp.5463-5467 (1977)). The whole or a part of the gene encodingthe desired protein can be obtained by excising the clone obtained asmentioned above with restriction enzymes and so on.

Also, the DNA encoding the desired protein can be isolated from thegenomic DNA derived from the cells expressing the desired protein asmentioned above by the following methods.

Such cells are solubilized preferably by SDS or proteinase K, and theDNAs are deproteinized by repeating phenol extraction. DNAs are digestedpreferably with ribonuclease. The DNAs obtained are partially digestedwith appropriate restriction enzymes, and the DNA fragments obtained areamplified with appropriate phage or cosmid to generate a library. Then,clones having the desired sequence are detected, for example, by usingradioactively labeled DNA probes, and the whole or a portion of the geneencoding the desired protein is obtained from the clones by excisionwith restriction enzymes etc.

A DNA encoding a desired protein can be prepared by following standardmethods using known mRNA or cDNA of the desired protein as a template bymeans of PCR (Gene Amplification PCR method, Basics and NovelDevelopment, Kyoritsu Publishers, 1992, etc).

A DNA encoding a desired protein can also be produced by chemicalsynthesis according to a usual method based on the nucleotide sequenceencoding the protein.

The rat CTGF of the present invention can be prepared as a recombinantprotein according to the frequently used recombinant technology by usingDNA obtained by digesting the rat CTGF-encoding DNA (the cDNA or thegenomic DNA comprising introns) prepared by the method indicated abovewith appropriate restriction enzymes; linking the resulting DNA fragmentencoding the rat CTGF, according to need, with a linker DNA or Tag byusing an appropriate DNA polymerase or other enzymes.

Specifically, the preparation of the protein is illustrated as follows:the DNA construct as prepared above is inserted into a vector, describedbelow in detail, to obtain an expression vector; a host cell, which willbe described hereinafter, is transformed with the expression vector toobtain a transformant; the resulting transformant cells are cultured forthe production and accumulation of the desired protein in the culturesupernatant; the protein accumulated in the culture supernatant can bepurified easily by using column chromatography, etc.

The present invention also relates to an expression vector comprisingthe DNA encoding the rat CTGF of the present invention. As an expressionvector of the present invention, any vector can be used as long as it iscapable of retaining replication or self-multiplication in each hostcell of prokaryotic and/or eukaryotic cells, including plasmid vectorsand phage vectors (Cloning Vectors: A laboratory Manual, Elsevier, N.Y.,1985).

The recombinant vector can easily be prepared by ligating the DNAencoding rat CTGF of the present invention with a vector forrecombination available in the art (plasmid DNA and bacteriophage DNA)by the usual method. Specific examples of the vectors for recombinationused are E. coli-derived plasmids such as pBR322, pBR325, pUC12, pUC13,and pUC19, yeast-derived plasmids such as pSH19 and pSH15, and Bacillussubtilis-derived plasmids such as pUB110, pTP5, and pC194. Examples ofphages are a bacteriophages such as λ phage, and an animal or insectvirus (pVL1393, Invitrogen) such as a retrovirus, vaccinia virus, andnuclear polyhedrosis virus.

A plasmid vector is useful for expressing the DNA encoding rat CTGF andfor producing rat CTGF. The plasmid vector is not limited as long as itexpresses the gene encoding the rat CTGF in various prokaryotic and/oreukaryotic host cells and produces this polypeptide. Examples thereofare pMAL C2, pcDNA3.1(−), pEF-BOS (Nucleic Acids Res. Vol.18, p.5322(1990) and so on), pME18S (Experimental Medicine: SUPPLEMENT, “Handbookof Genetic Engineering” (1992) and so on), etc.

When bacteria, particularly E. coli are used as host cells, anexpression vector is generally comprised of, at least, apromoter/operator region, an initiation codon, the DNA encoding theprotein of the present invention, termination codon, terminator region,and replicon.

When yeast, animal cells, or insect cells are used as hosts, anexpression vector is preferably comprised of, at least, a promoter, aninitiation codon, the DNA encoding the rat CTGF of the presentinvention, and a termination codon. It may also comprise the DNAencoding a signal peptide, enhancer sequence, 5′- and 3 ′-untranslatedregion of the gene encoding the rat CTGF of the present invention,splicing junctions, polyadenylation site, selectable marker region, andreplicon. The expression vector may also contain, if required, a genefor gene amplification (marker) that is usually used.

A promoter/operator region to express the fusion polypeptide of thepresent invention in bacteria comprises a promoter, an operator, and aShine-Dalgarno (SD) sequence (for example, AAGG). For example, when thehost is Escherichia, it preferably comprises Trp promoter, lac promoter,recA promoter, λPL promoter, 1pp promoter, tac promoter, or the like.

Examples of a promoter to express the rat CTGF of the present inventionin yeast are PH05 promoter, PGK promoter, GAP promoter, ADH promoter,and so on. When the host is Bacillus, examples thereof are SL01promoter, SP02 promoter, penP promoter and so on.

When the host is a eukaryotic cell such as a mammalian cell, examplesthereof are SV40-derived promoter, retrovirus promoter, heat shockpromoter, and soon, and preferably SV-40 and retrovirus-derived one. Asa matter of course, the promoter is not limited to the above examples.In addition, using an enhancer is effective for expression.

A preferable initiation codon is, for example, a methionine codon (ATG).

A commonly used termination codon (for example, TAG, TAA, TGA) isexemplified as a termination codon.

Usually, used natural or synthetic terminators are used as a terminatorregion.

A replicon means a DNA capable of replicating the whole DNA sequence inhost cells, and includes a natural plasmid, an artificially modifiedplasmid (DNA fragment prepared from a natural plasmid), a syntheticplasmid, and so on. Examples of preferable plasmids are pBR322 or itsartificial derivatives (DNA fragment obtained by treating pBR322 withappropriate restriction enzymes) for E. coli, yeast 2μ plasmid or yeastchromosomal DNA for yeast, and pRSVneo ATCC 37198, pSV2dhfr ATCC 37145,pdBPV-MMTneo ATCC 37224, pSV2neo ATCC 37149, pSV2bsr, and such formammalian cells.

An enhancer sequence, polyadenylation site, and splicing junction thatare usually used in the art, such as those derived from SV40 can also beused.

A selectable marker usually employed can be used according to the usualmethod. Examples thereof are resistance genes for antibiotics, such astetracycline, ampicillin, or kanamycin.

Examples of genes for gene amplification are dihydrofolate reductase(DHFR) gene, thymidine kinase gene, neomycin resistance gene, glutamatesynthase gene, adenosine deaminase gene, ornithine decarboxylase gene,hygromycin-B-phophotransferase gene, aspartate transcarbamylase gene,etc.

The expression vector of the present invention can be prepared bycontinuously and circularly linking at least the above-mentionedpromoter, initiation codon, DNA encoding the protein of the presentinvention, termination codon, and terminator region, to an appropriatereplicon. If desired, appropriate DNA fragments (for example, linkers,restriction sites generated with other restriction enzyme), can be usedby the usual method such as digestion with a restriction enzyme orligation using T4 DNA ligase.

Transformants of the present invention can be prepared by introducingthe expression vector mentioned above into host cells.

Host cells used in the present invention are not limited as long as theyare compatible with an expression vector mentioned above and can betransformed. Examples thereof are various cells such as wild-type cellsor artificially established recombinant cells usually used in technicalfield of the present invention (for example, bacteria (Escherichia andBacillus), yeast (Saccharomyces, Pichia, and such), animal cells, orinsect cells).

E. coli or animal cells are preferably used. Specific examples are E.coli (DH5 α, DH10B, TB1, HB101, XL-2Blue, and such), mouse-derived cells(COP, L, C127, Sp2/0, NS-1, NIH 3T3, and such), rat-derived cells,hamster-derived cells (BHK, CHO, and such), monkey-derived cells (COS1,COS3, COS7, CV1, Velo, and such), and human-derived cells (Hela, diploidfibroblast-derived cells, myeloma, Namalwa, and such).

An expression vector can be introduced (transformed (transduced)) intohost cells by known methods.

Transformation can be performed, for example, according to the method ofCohen et al. (Proc. Natl. Acad. Sci. USA, Vol.69, p.2110 (1972)),protoplast method (Mol. Gen. Genet., Vol.168, p.111 (1979)), orcompetent method (J. Mol. Biol., Vol.56, p.209 (1971)) when the hostsare bacteria (E. coli, Bacillus subtilis, and such), the method ofHinnen et al. (Proc. Natl. Acad. Sci. USA, Vol.75, p.1927 (1978)), orlithium method (J. Bacteriol., Vol.153, p.163 (1983)) when the host isSaccharomyces cerevisiae, the method of Graham (Virology, Vol.52, p.456(1973)) when the hosts are animal cells, and the method of Summers etal. (Mol. Cell. Biol., Vol.3, pp.2156-2165 (1983)) when the hosts areinsect cells.

Rat CTGF of the present invention can be produced by cultivatingtransformants (in the following this term includes transductants)comprising an expression vector prepared as mentioned above in nutrientmedia.

The nutrient media preferably comprise carbon source, inorganic nitrogensource, or organic nitrogen source necessary for the growth of hostcells (transformants). Examples of the carbon source are glucose,dextran, soluble starch, and sucrose, and examples of the inorganic ororganic nitrogen source are ammonium salts, nitrates, amino acids, cornsteep liquor, peptone, casein, meet extract, soy bean cake, and potatoextract. If desired, they may comprise other nutrients (for example, aninorganic salt (for example, calcium chloride, sodiumdihydrogenphosphate, and magnesium chloride), vitamins, antibiotics (forexample, tetracycline, neomycin, ampicillin, kanamycin, and so on).

Cultivation is performed by a method known in the art. Cultivationconditions such as temperature, pH of the media, and cultivation timeare selected appropriately so that the protein of the present inventionis produced in large quantities.

Specific media and cultivation conditions used depending on host cellsare illustrated below, but are not limited thereto.

When the hosts are bacteria, actinomycetes, yeasts, filamentous fungi,liquid media comprising the nutrient source mentioned above areappropriate. The media with pH 5 to 8 are preferably used. When the hostis E. coli, examples of preferable media are LB media, M9media(Milleretal. Exp. Mol. Genet., Cold Spring Harbor Laboratory, p.431(1972)), YT medium, and so on. Using these media, cultivation can beperformed usually at 14 to 43° C. for about 3 to 24 hours with aerationand stirring, if necessary.

When the host is Bacillus, cultivation can be performed usually at 30 to40° C. for about 16 to 96 hours with aeration and stirring, ifnecessary.

When the host is yeast, an example of media is Burkholder minimal media(Bostian, Proc. Natl. Acad. Sci. USA, Vol.77, p.4505 (1980)). The pH ofthe media is preferably 5 to 8. Cultivation can be performed usually at20 to 35° C. for about 14 to 144 hours with aeration and stirring, ifnecessary.

When the host is an animal cell, examples of media are MEM mediacontaining about 5 to 20% fetal bovine serum (Science, Vol.122, p.501(1952)), DMEM media (Virology, Vol.8, p.396 (1959)), RPMI1640 media (J.Am. Med. Assoc., Vol.199, p.519 (1967)), 199 media (Proc. Soc. Exp.Biol. Med., Vol.73, p.1 (1950)), HamF12 media, and so on. The pH of themedia is preferably about 6 to 8. Cultivation can be performed usuallyat about 30 to 40° C. for about 15 to 72 hours with aeration andstirring, if necessary.

When the host is an insect cell, an example of media is Grace's mediacontaining fetal bovine serum (Proc. Natl. Acad. Sci. USA, Vol.82,p.8404 (1985)). The pH thereof is preferably about 5 to 8. Cultivationcan be performed usually at about 20 to 40° C. for 15 to 100 hours withaeration and stirring, if necessary.

Rat CTGF of the present invention can be produced by cultivatingtransformants as mentioned above (in particular animal cells or E. coli)and allowing them to secrete the protein into the culture supernatant.Namely, a culture filtrate (supernatant) is obtained by a method such asfiltration or centrifugation of the obtained culture, and the rat CTGFof the present invention is purified and isolated from the culturefiltrate by methods commonly used in order to purify and isolate anatural or synthetic protein.

Examples of the isolation and purification method are a method utilizingaffinity, such as affinity column chromatography; a method utilizingsolubility, such as salting out and solvent precipitation method; amethod utilizing the difference in molecular weight, such as dialysis,ultrafiltration, gel filtration, and sodium dodecylsulfate-polyacrylamide gel electrophoresis; a method utilizing charges,such as ion exchange chromatography and hydroxylapatite chromatography;a method utilizing the difference in hydrophobicity, such as reversephase high performance liquid chromatography; and a method utilizing thedifference in isoelectric point, such as isoelectric focusing.

When the rat CTGF of the present invention exists in the periplasm orcytoplasm of cultured transformants, first, the cells are harvested by ausual method such as filtration or centrifugation and suspended inappropriate buffer. After the cell wall and/or cell membrane of thecells and such are disrupted by the method such as lysis withsonication, lysozyme, and freeze-thawing, the membrane fractioncomprising the rat CTGF of the present invention is obtained by themethod such as centrifugation or filtration. The membrane fraction issolubilized with a detergent such as Triton-X100 to obtain the crudeextract. Finally, the protein is isolated and purified from the crudeextract by the usual method as illustrated above.

The “transgenic mouse” of the present is a transgenic mouse in which theabove human CTGF encoding DNA (cDNA or genomic DNA) prepared by themethod mentioned above has been integrated into the endogenous genelocus of the mouse. This transgenic mouse expresses and secretes thehuman CTGF in vivo.

The transgenic mouse can be prepared according to the method usuallyused for producing a transgenic animal (for example, see “Newest Manualof Animal Cell Experiment”, LIC press, Chapter 7, pp.361-408, (1990)).Specifically, for example, a transgenic mouse can be produced asfollows. Embryonic stem cells (ES cells) obtained from normal mouseblastocysts are transformed with an expression vector in which the geneencoding the human CTGF has been inserted in an expressible manner. EScells in which the gene encoding the human CTGF has been integrated intothe endogenous gene are screened by a usual method. Then, the ES cellsscreened are microinjected into a fertilized egg (blastocyst) obtainedfrom another normal mouse (Proc. Natl. Acad. Sci. USA, Vol.77, No.12,pp.7380-7384 (1980); U.S. Pat. No. 4,873,191). The blastocyst istransplanted into the uterus of another normal mouse as the fostermother and chimeric transgenic mice are born. By mating the chimerictransgenic mice with normal mice, heterozygous transgenic mice areobtained. By mating the heterozygous transgenic mice with each other,homozygous transgenic mice are obtained according to Mendel's laws.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the properties of various monoclonal antibodies prepared byimmunizing a variety of mammals with human CTGF or mouse CTGF.

FIG. 2 shows the properties of various human monoclonal antibodiesprepared by immunizing human antibody-producing transgenic mice withhuman CTGF.

FIG. 3 is an SDS-polyacrylamide gel electrophoretic pattern of human,mouse, and rat recombinant CTGFs. The proteins were affinity purified byusing a column coupled with the monoclonal antibody “8-86-2” reactive toall of human, mouse and rat CTGFS.

FIG. 4 shows the growth-promoting activities of human, mouse, and ratCTGFs for the rat kidney-derived fibroblast cell line NRK-49F. Theproteins were affinity purified by using a column coupled with themonoclonal antibody “8-86-2” reactive to all of human, mouse, and ratCTGFs.

The ordinate indicates the [³H]-thymidine uptake of the cells as anindex of the growth promoting activity; the abscissa indicates theconcentrations of the respective recombinant CTGFs used.

FIG. 5 shows the reactivity of various monoclonal antibodies to humanCTGF. The monoclonal antibodies were prepared by immunizing a variety ofmammals with human or mouse CTGF.

The ordinate indicates the fluorescence intensity as an index of thereactivity of the monoclonal; the abscissa indicates clone names of themonoclonal antibodies tested by ELISA with different concentrations ofthe human CTGF.

FIG. 6 shows the reactivity of various monoclonal antibodies to themouse CTGF. The monoclonal antibodies were prepared by immunizing avariety of mammals with human or mouse CTGF.

The ordinate indicates the fluorescence intensity as an index of thereactivity of the monoclonal antibody; the abscissa indicates clonenames of the monoclonal antibodies tested by ELISA with differentconcentrations of mouse CTGF.

FIG. 7 shows the reactivity of various monoclonal antibodies to ratCTGF. The monoclonal antibodies were prepared by immunizing a variety ofmammals with human or mouse CTGF.

The ordinate indicates the fluorescence intensity as an index of thereactivity of the monoclonal antibody; the abscissa indicates clonenames of the monoclonal antibodies tested by ELISA with differentconcentrations of rat CTGF.

FIG. 8 shows the reactivity of various human monoclonal antibodies tohuman CTGF. The antibodies were prepared by immunizing the humanantibody-producing transgenic mice with human CTGF.

The ordinate indicates the fluorescence intensity as an index of thereactivity of the monoclonal antibody; the abscissa indicates clonenames of the human monoclonal antibodies tested by ELISA with differentconcentrations of human CTGF.

FIG. 9 shows the reactivity of various human monoclonal antibodies tomouse CTGF. The antibodies were prepared by immunizing the humanantibody-producing transgenic mice with human CTGF.

The ordinate indicates the fluorescence intensity as an index of thereactivity of the monoclonal antibody; the abscissa indicates clonenames of the human monoclonal antibodies tested by ELISA with differentconcentrations of mouse CTGF.

FIG. 10 shows the reactivity of various human monoclonal antibodies torat CTGF. The antibodies were prepared by immunizing the humanantibody-producing transgenic mice with human CTGF.

The ordinate indicates the fluorescence intensity as an index of thereactivity of the monoclonal antibody; the abscissa indicates clonenames of the human monoclonal antibodies tested by ELISA with differentconcentrations of rat CTGF.

FIG. 11 shows the inhibiting activity of various monoclonal antibodiesfor the binding of human or mouse CTGF to the human kidney-derivedfibroblast cell line 293-T. The monoclonal antibodies were prepared byimmunizing a variety of mammals with human or mouse CTGF.

The ordinate indicates that fluorescence intensity as an index of theinhibiting activity; the abscissa indicates clone names of variousmonoclonal antibodies tested.

FIG. 12 shows the inhibiting activity of various human monoclonalantibodies for the binding of human CTGF to the rat kidney-derivedfibroblast cell line NRK-49F. The antibodies were prepared by immunizinghuman antibody-producing transgenic mice with human CTGF.

The ordinate indicates the rate of bound cells (%); the abscissaindicates clone names of the various monoclonal antibodies tested. Thetotal number of cells added is taken as 100%.

FIG. 13 shows the inhibiting activity of various human monoclonalantibodies for the binding of human CTGF to the rat kidney-derivedfibroblast cell line NRK-49F, the human osteosarcoma-derived cell lineMG-63, or human lung-derived fibroblast cells. The antibodies wereprepared by immunizing the human antibody-producing transgenic mice withhuman CTGF.

The ordinate indicates the rate of bound cells (%); the abscissaindicates clone names of the various monoclonal antibodies tested. Thetotal number of cells added is taken as 100%.

FIG. 14 shows immunological staining patterns of tissue sections fromarteriosclerotic lesions of a rabbit as an arteriosclerosis modelrabbit. By using various monoclonal antibodies prepared by immunizing avariety of mammals with human or mouse CTGF, the sections were stainedand the reactivity of the antibodies to the lesions was assessed.

The panel (a) shows a control stain; (b) shows the stain with themonoclonal antibody B35.1; (c) shows the stain with the monoclonalantibody B29.6; (d) shows the stain with the monoclonal antibody13-51-2; (e) shows the stain with the monoclonal antibody A4.3; (f)shows the stain with the monoclonal antibody C114.4; (g) shows the stainwith the monoclonal antibody A11.1; (h) shows the stain with themonoclonal antibody A29.6; and (1) shows the stain with the monoclonalantibody C26.11.

FIG. 15 shows the inhibiting activity of various human monoclonalantibodies towards for the proliferation of the rat kidney-derivedfibroblast cells NRK-49F stimulated by the purified human CTGF. Theantibodies were prepared by immunizing the human antibody-producingtransgenic mice with human CTGF.

The ordinate indicates the [³H]-thymidine uptake of the cells as anindex of the growth promoting activity; the abscissa indicates clonenames of the human monoclonal antibodies tested by using the purifiedhuman CTGF of different concentrations.

FIG. 16 shows the inhibiting activity of various human monoclonalantibodies towards the proliferation of the rat kidney-derivedfibroblast cells NRK-49F stimulated by the purified human CTGF. Theantibodies were prepared by immunizing the human antibody-producingtransgenic mice with human CTGF.

The ordinate indicates the [³H]-thymidine uptake of the cells as anindex of the growth promoting activity; the abscissa indicates clonenames of the human monoclonal antibodies tested by using purified humanCTGF of different concentrations.

FIG. 17 shows the therapeutic effect of various human monoclonalantibodies on kidney diseases and fibrotic diseases in tissues. Theantibodies were prepared by immunizing the human antibody-producingtransgenic mice with human CTGF.

The ordinate indicates the concentration of hydroxyproline which is anindex of advancement of the disease; the abscissa indicates clone namesof the human monoclonal antibodies administered.

FIG. 18 schematically shows the procedure for the sequence determinationof the DNAs encoding the heavy chain and the light chain of the humananti-human CTGF monoclonal antibody.

FIG. 19 shows the calibration curves of the human CTGF standard, mouseCTGF standard, and rat CTGF standard. The curves were obtained bysandwich ELISA using the monoclonal antibodies 8-64-6 and 8-86-2.

The ordinate indicates the fluorescence intensity; the abscissaindicates the concentration of the standards of CTGF.

FIG. 20 shows the calibration curves of the human CTGF standard, mouseCTGF standard, and rat CTGF standard. The curves were obtained bysandwich ELISA using the monoclonal antibodies 13-51-2 and 8-86-2.

The ordinate indicates the fluorescence intensity; the abscissaindicates the concentration of the standards of CTGF.

FIG. 21 shows CTGF concentrations in various serum samples from patientsaffected with biliary atresia. The concentration was determined bysandwich ELISA with the monoclonal antibodies 8-64-6 and 8-86-2.

The ordinate indicates the concentrations (CTGF content) determined; theabscissa indicates the subject groups tested. The samples were obtainedfrom three groups; Group 1 (I) consisting of patients with normalclinical findings; Group 2 (II) consisting of patients with symptomsprogressing; and Group 3 (III) consisting of patients with severesymptoms in need of liver transplantation.

FIG. 22 shows CTGF concentrations in various serum samples from patientsaffected various diseases. The concentration was determined by sandwichELISA with the monoclonal antibodies 8-64-6 and 8-86-2.

The ordinate indicates the concentrations (CTGF content) determined; theabscissa indicates the diseases with which the patients affected.

FIG. 23 shows CTGF concentrations in the synovial fluid samples frompatients affected with rheumatoid arthritis or osteoarthritis. Theconcentration was determined by sandwich ELISA with the monoclonalantibodies 8-64-6 and 8-86-2.

The ordinate indicates the concentrations (CTGF content) determined; theabscissa indicates the diseases with which the patients affected.

FIG. 24 are SDS-polyacrylamide gel patterns showing the result ofWestern blotting of human CTGF fractions purified from human fetal 25skin-derived fibroblast cells by using a heparin column.

Lanes 1, 2, and 3 contain fractions eluted with 0.2 M, 0.6 M, and 2.0MNaCl, respectively, and the samples were treated with pre-immuneantibody (pre-immune serum from normal rabbit). Lanes 4, 5, and 6contain the fractions eluted with 0.2 M, 0.6 M, and 2.0M NaCl,respectively, and the samples were treated with an anti-human CTGFpolyclonal antibody.

FIG. 25 shows the growth-promoting activity of human CTGF for ratkidney-derived fibroblast cells NRK-49F. The human CTGF is purified fromhuman fetal skin-derived fibroblast cells by using a heparin column. Thefraction eluted with 0.6M NaCl was used after diluting 10-, 30-, 100-,or 300 times.

“PDGF” denotes the PDGF used as a positive control in this assay; “NC”indicates negative control where the CTGF was omitted in the assay.

REST MODE FOR CARRYING OUT THE INVENTION

The present inventions are further described in detail by referring toExamples herein below, but are not to be construed as being restrictedthereto.

EXAMPLE 1 Preparation of a Polyclonal Antibody to Human CTGF

The peptide corresponding to amino acid residues in the positions of 242to 252 of human CTGF was synthesized with a peptide synthesizer (AppliedBiosystems) according to a usual method. The peptide emulsified withFreund's complete adjuvant was used as an immunogen. The peptide (0.32mg/kg) was given subcutaneously to a New Zealand white rabbit (NZW;Simunek, Inc.). The dose and the interval were: 0.8 mg of the peptide onday 1; 0.8 mg on day 14; 0.8 mg on day 35; and 0.8 mg on day 49. Theantibody titer in the serum was assayed from time to time by using thepeptide. The serum was then collected according to a usual method. Thepolyclonal antibody (IgG) against human CTGF was purified from the serumby affinity chromatography using agarose on which the peptide wasimmobilized. The reactivity to human CTGF was verified by Enzyme-linkedimmunosorbent assay (ELISA ) and Western blotting.

EXAMPLE 2 Preparation of Recombinant Human CTGF

<2-1> The transient Expression of Human Recombinant CTGF in HumanKidney-derived Fibroblast Cell Line 293

Complementary DNA encoding human CTGF was prepared according to a usualmethod by PCR. Specifically, the cDNA was prepared by using, as atemplate, heterogeneous cDNAs derived from a human chondroma cell line,HCS2/8, and by using primers designed based on the human CTGF cDNAsequence (The Journal of Cell Biology, Vol. 114, No. 6, p. 1287-1294,1991). The human CTGF cDNA so obtained comprising the coding region wasinserted into a plasmid, pcDNA3.1 (−) (Invitrogen Co.) to construct theexpression vector. The human kidney-derived fibroblast cell line 293-T(ATCC CRT1573) was transformed with the prepared vector byelectroporation. The transformant was cultured in a serum-free medium,ASF104 (Ajinomoto Co. Inc.), for three days, for the transientexpression of the recombinant human CTGF. The human CTGF expression wasconfirmed by Western blotting using the polyclonal antibody prepared inExample 1.

The collected culture supernatant was subjected to salting-out withammonium sulfate, and then to heparin-column chromatography. The columnwas washed with 0.3M NaCl/PBS and the human CTGF fraction was elutedwith 0.5M NaCl/PBS. Thus, the partially purified human CTGF wasobtained.

<2-2> Stable Expression of Human Recombinant CTGF in a Human EpithelioidCell Line, Hela Cell

Complementary DNA encoding human CTGF was prepared according to a usualmethod by PCR in the same manner as described in Example <2-1>. Thehuman CTGF cDNA so obtained comprising the coding region was insertedinto a plasmid, pcDNA3.1 (−) (Invitrogen Co.) to construct theexpression vector. The human epithelioid cell line, Hela cell (ATCCCCL-2), was transformed with the prepared vector by electroporation. Thetransformants were cultured in a RPMI1640 medium containing GENETICIN(0.8 mg/ml; GIBCO-BRL) and 10% fetal calf serum for about two weeks, inorder to select GENETICIN-resistant clones of the transformant. Thetransformant selected was cultured in the serum-free medium ASF104(Ajinomoto Co. Inc.) for the expression of the recombinant human CTGF.The expression of the human CTGF was verified by Western blotting usingthe polyclonal antibody prepared in Example 1.

The collected culture supernatant was subjected to salting-out 30 withammonium sulfate, and then to heparin-column chromatography. The columnwas washed with 0.3M NaCl/PBS and the human CTGF fraction was elutedwith 0.5M NaCl/PBS. Thus, the partially purified human CTGF wasobtained.

EXAMPLE 3 Preparation of Recombinant Mouse CTGF

Partially purified recombinant mouse CTGF was prepared by the samemethod described in Example 2, based on the CDNA sequence of mouse CTGFreported previously (Unexamined Published Japanese Patent Application(JP-A) No. Hei 5-255397; Cell Growth Differ., Vol. 2, No. 5, p. 225-233,1991; FEBS Letters, Vol. 327, No. 2, p. 125-130, 1993; DNA Cell Biol.,Vol. 10, No. 4, p. 293-300, 1991).

EXAMPLE 4 Preparation of Anti-human CTGF Monoclonal Antibody andAnti-mouse CTGF Monoclonal Antibody

Preparation of the monoclonal antibodies in this example was performedby the conventional method described in “Experimental Medicine(supplement): Handbook for Cellular Engineering Technology, eds., T.Kuroki et al., Yodosha, page 66-74, 1992),” and “Introductory Manual forMonoclonal Antibody Experiment (T. Ando et al., Kodansha, 1991).”

Here in this example, either of the recombinant human CTGF preparationsobtained by either of the two methods described in Example 2, or amixture thereof, was used as an immunogen. The mouse CTGF used was therecombinant mouse CTGF prepared in Example 3.

The immune animals used were: (1) normal mouse (Balb/c mouse, female, 4-to 5-week old; Shizuoka experimental animal center); (2) normal rat(Wistar rat, female, 4- to 5-week old; Shizuoka experimental animalcenter); (3) normal hamster (Armenian hamster, male, 4- to 5-week-old;Oriental Yeast Co., Ltd.); and (4) the human antibody-producingtransgenic mouse created by using the method described above (refer thefollowing reports: Nature Genetics, Vol. 7, p. 13-21, 1994; NatureGenetics, Vol. 15, p. 146-156, 1997; Published Japanese Translation ofPCT International Application No. Hei 4-504365; Published JapaneseTranslation of PCT International Application No. Hei 7-509137; NikkeiScience, June edition, page 40-50, 1995).

Unless otherwise stated, the same method was used for the preparation ofmonoclonal antibodies derived from any animal. Multi-well microplateswere used for culturing cells.

<4-1> Preparation of Hybridomas Producing Anti-human CTGF MonoclonalAntibody

The above-mentioned normal mouse and the human-antibody producingtransgenic mouse were immunized with the partially purified recombinanthuman CTGF (1 μg/animal) prepared in Example 2. The immunogen, togetherwith complete Freund's adjuvant, was given to the mice by footpadinjection for primary immunization (day 0). The recombinant human CTGFwas given to the mice by footpad injection every week after the primaryimmunization. The booster immunization was performed four times or morein total. The final immunization was carried out by the same proceduretwo days before the collection of lymph node cells describedhereinafter.

The lymph node cells collected from each animal and mouse myeloma cellswere mixed at a ratio of 5:1. Hybridomas were prepared by cell fusionusing polyethylene glycol 4000 or polyethylene glycol 1500 (GIBCO) as afusing agent. The lymph node cells of the normal mouse were fused withmouse myeloma PAI cells (JCR No. B0113; Res. Disclosure Vol. 217, p.155, 1982), and the lymph node cells of human antibodyproducing-transgenic mouse were fused with mouse myeloma P3/X63-AG8.653cells (ATCC No. CRL 1580).

The resulting hybridomas were selected by culturing the fused cells inan ASF104 medium (Ajinomoto Co. Inc.) containing HAT supplemented with10% fetal calf serum (FCS) and aminopterin.

The reactivity of the culture supernatant of each hybridoma clone to therecombinant human CTGF used as the immunogen was measured by ELISAdescribed hereinafter. Many antibody producing-hybridomas were thusobtained from each animal species.

The clones named 8-64-6, 8-86-2, 8-97-3, 8-149-3, and 15-38-1 (FIG. 1)were obtained from normal mice (mouse anti-human CTGF monoclonalantibodies).

The hybridoma clones, 8-86-2 and 8-64-6, both have been depositedinternationally since Dec. 18, 1997 at The National Institute ofBioscience and Human-Technology, The Agency of Industrial Science andTechnology, The Ministry of International Trade and Industry(1-1-3Higashi, Tsukuba, Ibaraki, Japan) (clone 8-86-2: internationaldeposit accession No. FERM BP-6208; clone 8-64-6: international depositaccession No. FERM BP-6209).

The clones (producing the human anti-human CTGF monoclonal antibodies)named A4.3, A11.1, A15.1, A29.6, B13.7, B22.2, B29.6, B35.1, C2.1,C26.11, C59.1, C114.4, M32.2, M33.3, M84.4, M107.2, M122, M124.6,M194.2, M244, M255, M268.1, M288.5, M291.2, M295.2, M315, M320.2, N45.2,N50.1, and N60.1 (FIGS. 1 and 2) were obtained from the humanantibody-producing transgenic mice.

The hybridoma clone A11.1 has been deposited internationally since Sep.25, 1998 at The National Institute of Bioscience and Human-Technology,The Agency of Industrial Science and Technology, The Ministry ofInternational Trade and Industry (1-1-3 Higashi, Tsukuba, Ibaraki,Japa-n)(international deposit accession No. FERM BP-6535).

The hybridoma clones, B22.2, M84.4, and M320.2 have been depositedinternationally since Dec. 15, 1998 at The National Institute ofBioscience and Human-Technology, The Agency of Industrial Science andTechnology, The Ministry of International Trade and Industry (1-1-3Higashi, Tsukuba, Ibaraki, Japan) (clone B22.2: international depositaccession No. FERM BP-6598; clone M84.4: international deposit accessionNo. FERM BP-6599; clone M320.2: international deposit accession No. FERMBP-6600).

Described above, the hybridoma clones producing the human monoclonalantibodies of the present invention are indicated by symbols all throughthe examples including the present example and the drawings or thetables showing the experimental results obtained in each example.

The alphabet followed by numerals up to the dot mark of each symbolcorresponds to the name of parental clone. The numerals after the dotmark of the symbol means a subclone obtained from the parental clone bysubcloning.

The numerals indicating the subclones may occasionally be abbreviated inany of the examples including the present example and the drawings orthe tables showing the experimental results obtained in each example.However, it should be noted that the abbreviated symbols indicate thesame clones as those indicated by the non-abbreviated symbols in FIGS. 1and 2.

<4-2> Preparation of Hybridomas Producing Anti-mouse CTGF MonoclonalAntibody

The above-mentioned normal rat and normal hamster were immunized withpartially purified recombinant mouse CTGF (2 μg/animal) prepared inExample 3, together with complete Freund's adjuvant, by footpadinjection for primary immunization (day 0). The booster immunization wasperformed by footpad injection every week after the primary immunizationfour times or more in total. The final immunization was carried out bythe same procedure two days before the collection of lymph node cells.The collection of lymph node cells is described below.

Popliteal lymph node cells were collected from each immunized animalsaccording to a usual method by a surgical operation.

The lymph node cells collected from each animal and myeloma cells PAI(JCR No.B0113; Res. Disclosure Vol. 217, p. 155, 1982) were mixed at aratio of 5:1. The hybridomas were prepared by cell fusion usingpolyethylene glycol 4000 or polyethylene glycol 1500 (GIBCO) as a fusingagent.

The hybridomas were selected by culturing the fused cells in an ASF104medium (Ajinomoto Co. Inc.) containing HAT supplemented with 10% fetalcalf serum (FCS) and aminopterin.

The reactivity of the culture supernatant of each hybridoma to therecombinant mouse CTGF used as the immunogen was measured by ELISAdescribed hereinafter. Many antibody producing-hybridomas were, thus,obtained from each animal.

The clones (producing rat anti-mouse CTGF monoclonal antibodies) named13-51-2, 17-132, 23-96, 24-53, 24-67, 25-91, 25-101, 25-256, 25-338,25-410, and 25-463 (FIG. 1) were obtained from normal rats.

The clone (producing hamster anti-mouse CTGF monoclonal antibody) named2-228-1 (FIG. 1) was obtained from normal hamster.

<4-3> Screening of Hybridomas Producing Monoclonal Antibodies by ELISA

ELISA performed in Examples <4-1> and <4-2> is as follows.

The recombinant human CTGF (0.2 μg/well) prepared in Example 2 or therecombinant mouse CTGF (0.2 μg/well) prepared in Example 3 was addedinto each well of a 96-well ELISA microplate (Corning Costar Co.). Theplate was incubated at room temperature for 2 hours for the adsorptionof the recombinant human CTGF or the recombinant mouse CTGF onto themicroplate. The supernatants were discarded and then the blockingreagent (200 μl; phosphate buffer containing 3% BSA) was added into eachwell. The plate was incubated at room temperature for 2 hours for theblocking of CTGF-free sites on the microplate.

Each well was washed three times with 200 μl of phosphate buffercontaining 0.1% Tween 20. Thus, each well of the microplate was coatedwith the recombinant human CTGF or recombinant mouse CTGF.

Culture supernatant (100 μl) of each hybridoma was added into each wellof the plate, and the reaction was allowed to proceed for 40 minutes.Each well was then washed three times with 200 μl of phosphate buffercontaining 0.1% Tween 20.

In the next step, biotin-labeled sheep anti-mouse immunoglobulinantibody (50 μl; Amersham) was added to the wells where the culturesupernatant of the monoclonal antibody-producing hybridoma derived fromnormal mouse had been placed; biotin-labeled sheep anti-ratimmunoglobulin antibody (50 μl; Amersham) was added to the wells wherethe culture supernatant of the monoclonal antibody-producing hybridomaderived from normal rat had been placed; biotin-labeled goatanti-hamster immunoglobulin antibody (50 μl; Cedarlane) was added to thewells where the culture supernatant of the monoclonal antibody-producinghybridoma derived from normal hamster had been placed; biotin-labeledgoat anti-human immunoglobulin antibody (50 μl; American QualexInternational Inc.) was added to the wells where the culture supernatantof the monoclonal antibody-producing hybridoma derived from the humanantibody-producing transgenic mouse had been placed. The plates wereincubated at room temperature for 1 hour.

The microplate was washed with phosphate buffer containing 0.1% Tween20. A solution of streptavidin-β-galactosidase (50 μl; Gibco-BRL),diluted 1000 times with a solution (pH7.0) containing 20 mM HEPES, 0.5MNaCl and bovine serum albumin (BSA, 1 mg/ml), was added into each well.The plate was incubated at room temperature for 30 minutes.

Subsequently, the microplate was washed with phosphate buffer containing0.1% Tween 20. A solution of 1% 4-Methyl-umbelliferyl-β-D-galactoside(50 μl; Sigma) in a phosphate buffer (pH7.0) containing 100 mM NaCl, 1mM MgCl₂ and 1 mg/ml BSA, was added into each well. The plate wasincubated at room temperature for 10 minutes. 1M Na₂CO₃ (100 μl) wasadded into each well to stop the reaction. The fluorescence intensitywas measured in a FlUOROSCAN II MICROPLATE FLUOROMETER (FlowLaboratories Inc.) at a wavelength of 460 nm (excitation wavelength: 355nm).

<4-4> Large-scale Preparation of Monoclonal Antibodies

Each hybridoma clone (10⁶-10⁷ cells/0.5 ml/each mouse) described abovewas injected intraperitoneally to ICR nude mice (female, 7- to 8-weekold; Charles River). Ten to twenty days after the injection, the asciticfluids were collected from the mice according to a usual method byopening the abdomen under anesthesia. The monoclonal antibodies wereprepared from the ascitic fluids in a large quantity.

<4-5> Purification of Monoclonal Antibodies

Each monoclonal antibody-containing ascitic fluid obtained in <4-4> wascentrifuged, and the resulting supernatant was diluted 3 times with0.06M acetate buffer (pH 4.0). The pH of the dilute was adjusted to 4.8by adding IN hydrochloric acid. Subsequently, 0.033 ml of caprylic acid(Wako Pure Chemical Industries. Ltd.) was added little by little toevery 1 ml of the ascitic fluid, while stirring the mixture at roomtemperature. The mixture was allowed to react for 30 minutes, whilebeing stirred. The proteins, except the antibody, were removed bycentrifugation (10,000 rpm, for 20 minutes). The supernatant obtained bycentrifugation was filtered by using a filter (Millipore Co.), the whiteprecipitate was discarded. The filtrate obtained was dialyzed withphosphate buffer (for 2 hours).

After the dialysis, ammonium sulfate (26.2 g/100 ml) was added theretolittle by little, while stirring the mixture at room temperature. Thereaction of the mixture was carried out at 4° C. for 120 minutes, whilebeing stirred. The resulting precipitate was then recovered bycentrifugation (10,000 rpm, for 20 minutes). The recovered precipitatewas dissolved with phosphate buffer and dialyzed with phosphate buffer(at 4° C., for 24 hours). Each monoclonal antibody was thus purified.<4-6> Determination of the isotype

Each isotype of the above-mentioned anti-human CTGF monoclonal antibodyderived from normal mouse (8-64-6, 8-86-2, 8-97-3, 8-149-3, and 15-38-1)was determined by using an isotype-determining kit for mouse monoclonalantibody (Amersham). The determination was performed according to thesupplier's protocol attached to the kit. All the antibodies weredetermined to be IgGl/κ (FIG. 1).

Each isotype of the above-mentioned anti-human CTGF monoclonal antibodyderived from the human antibody-producing transgenic mouse (A4.3, A11.1,A15.1, A29.6, B13.7, B22.2, B29.6, B35.1, C2.1, C26.11, C59.1, C114.4,M32.2, M33.3, M84.4, M107.2, M122, M124.6, M194.2, M244, M255, M268.1,M288.5, M291.2, M295.2, M315, M320.2, N45.2, N50.1, and N60.1) wasdetermined by using an isotype-determining kit for human monoclonalantibody (American Qualex International Inc.). The determination wasperformed according to the supplier's protocol attached to the kit. Allthe antibodies were determined to be IgG2/κ (FIGS. 1 and 2).

<4-7> Preparation of the Affinity Column

An affinity column was prepared by using NHS-activated HiTrap column(HITRAP-NHS-ACTIVATED SEPHAROSE HP; 5 ml; Pharmacia Biotec.) accordingto the protocol attached to the product. Specifically, the preparationwas done as follows:

The monoclonal antibody 8-86-2 (10 mg/ml SEPHAROSE) prepared in Example<4-5> was dissolved in a 0.2M sodium hydrogen carbonate solution (pH8.3)containing 0.5M NaCl. The solution (10 mg/ml SEPHAROSE) was loaded ontothe NHS-ACTIVATED HITRAP COLUMN. The monoclonal antibody 8-86-2 wasallowed to react with the NHS-ACTIVATED SEPHAROSE at 20° C. for 45minutes, to immobilize the antibody on the SEPHAROSE.

Because of the high reactivity of the monoclonal antibody 8-86-2 tohuman, mouse and rat CTGFs, the affinity column prepared with theantibody can be used for purifying any human CTGF, mouse CTGF, and ratCTGF.

<4-8> Purification of Mammalian CTGF by Affinity Chromatography

Culture supernatant was collected from each culture of the HeLatransformant cells expressing human CTGF (Example <2-2>), the HeLatransformant cells expressing mouse CTGF (Example 3) and the HeLatransformant cells expressing rat CTGF (Example <11-2>). The supernatantwas fractionated by heparin-column chromatography. The column was washedwith 0.3M NaCl/PBS, and then the protein fraction of interest was elutedwith 0.7M NaCl/PBS. Thus, the crude fractions of human, mouse and ratCTGFs were obtained.

Each crude fraction was loaded onto the affinity column, prepared inExample <4-7>, in which the anti-CTGF antibody 8-86-2 had beenimmobilized. The column was washed with phosphate buffer. The fractionof interest was then eluted with 0.1M glycine buffer (pH2.5) and thenwas neutralized with 0.75M Tris-HCl buffer (pH9.0). The eluted fractionswere dialyzed against phosphate buffer. Thus, highly purifiedrecombinant CTGFs derived from human, mouse and rat were obtained.

The purified recombinant CTGFs were electrophoresed on a sodiumdodecylsulfate polyacrylamide gel with a concentration gradient of 10 to20% polyacrylamide. The separated bands on the gel were silver-stained,and bands of about 38-kDa corresponding to human, mouse and rat CTGFswere found on the stained gel (FIG. 3).

<4-9> Examination of the Stimulatory Activity of Purified CTGF for CellProliferation

To verify whether or not each of the CTGFs purified in Example <4-8> hasa biological activity, the stimulatory activity of purified CTGF forcell proliferation was tested.

The cells of rat kidney fibroblast cells NRK-49F (ATCC CRL-1570; 2×10³cells/well) were cultured in a DMEM medium containing 10% fetal calfserum (FCS) in a 96-well microplate for three days. The culturesupernatant was removed and the cells were washed once with the DMEMmedium. Then the cells were cultured in a fresh DMEM medium for one day.Subsequently, the purified recombinant CTGF was added in variousconcentrations (100, 50, 25, 12.5, 6.3, and 3.1 ng/ml) into each cellculture. The culture was continued for 2 days and then [³H]-thymidine(3.7kBq/well) was added thereto. After a 6-hour culture, the cells wereharvested for the measurement of [³H]-thymidine uptake by the cells. Themeasurement was carried out in a liquid scintillation counter (Beckman).The cells were cultured in the same manner but in the absence of CTGF,and [³H]-thymidine uptake by the cells was measured as a control.

The result is shown in FIG. 4. It was evidenced that each of thepurified recombinant CTGFS derived from human, mouse, and rat exhibiteda concentration-dependent stimulatory activity for the cellproliferation and accordingly all the recombinant CTGFs possessed abiological function.

<4-10> Crossreactivity

The reactivities of various anti-human CTGF monoclonal antibodies (10μg/ml) and anti-mouse CTGF monoclonal antibodies (10 μg/ml) to each ofhuman CTGF, mouse CTGF and rat CTGF were tested by ELISA in the samemanner described in Example <4-3>.

The microplates used in this assay were coated with each of therecombinant human, mouse and rat CTGFs purified by using affinity columnprepared in Example <4-7>. The coating was performed with the proteinsin the concentrations of (A) 100, 30, and 10 ng/well or (B) 100, 10, and1 ng/well.

In the assay of (B), negative control assay was carried out by using ahuman monoclonal antibody against KLH (keyhole limpet hemocyanin; PierceChemical Co.) in the same manner described above. The anti-KLH antibodywas prepared by immunizing the above-described human antibody-producingtransgenic mice with KLH as an immunogen.

The assay results obtained with the concentration series (A) are shownin FIGS. 5-7; the assay results obtained with the concentration series(B) are shown in FIGS. 8-10.

The results shown in FIGS. 5-10 are also summarized in the column of“crossreactivity” in FIGS. 1 and 2.

Within the column of “crossreactivity” of FIG. 1, the results are shownin the order of 100, 30 and 10 ng/well of the coating concentration fromthe left. In each coating concentration, the reactivity represented bythe fluorescence intensity of 1000 or more is marked with “◯”; 500 ormore and less than 1000, “Δ”; less than 500, “X.”

Within the column of “crossreactivity” of FIG. 2, the results are shownin the order of 100, 10 and 1 ng/well of the coating concentration fromthe left. In each coating concentration, the reactivity represented bythe fluorescence intensity of 1000 or more is marked with “◯”; 500 ormore and less than 1000, “Δ”; less than 500, “X.”

Thus, it was revealed that the monoclonal antibodies of the presentinvention had a variety of characteristics in terms of thecrossreactivity.

<4-11> Activity of Inhibiting the Binding of CTGF to Various Cells

A recent study has revealed that CTGF is involved in cell adhesion (Exp.Cell. Res., Vol. 233, p. 63-77, 1997). The inhibiting effect of thevarious monoclonal antibodies prepared above on the CTGF-mediated celladhesion was investigated as an index of judging whether or not theantibodies have the activity (neutralizing activity) of inhibiting theCTGF functions. The test was performed by using the three distinctmethods described below in <4-11-1> to <4-11-3>.

<4-11-1> Inhibition of the Binding of CTGF with Human Kidney-derivedFibroblast Cell Line 293-T

The above-prepared various monoclonal antibodies (0.5 μg/well) reactiveto human CTGF were added into the wells of a microplate coated with therecombinant human CTGF (the coating concentration: 0.5 μg/well). Theimmobilization of the human CTGF on the plate was performed by the sameprocedure described in Example <4-3>. The above-prepared variousmonoclonal antibodies (0.5 μg/well) reactive to mouse CTGF were addedinto the wells of a microplate coated with the recombinant mouse CTGF(the coating concentration: 0. 5 μg/well). The immobilization of themouse CTGF on the plate was performed by the same procedure described inExample <4-3>.

The supernatants were removed from each plate, and then the cells ofhuman kidney-derived fibroblast cell line 293-T (ATCC CRL1573) labeledwith 2′,7′-bis(2-carboxyethyl)-5(6)-carboxyfluoresceintetraacetoxymethyl ester (BCECF; Molecular Probes Inc.) were added intoeach well (5×10⁴ cells/well). The plates were allowed to stand at 4° C.for 1 hour.

The floating cells were removed and then the cells adhering to theplates were solubilized by adding phosphate buffer (100 μl) containing1% NP-40 thereto. The release of BCECF into the culture supernatant wascaused by cytolysis. The intensity of fluorescence emitted by BCECF wasmeasured by the FLUOROSCAN II MICROPLATE FLUOROMETER (Flow LaboratoriesInc.).

Control assay was carried out in the same manner described above butwithout any antibodies added.

The results are shown in FIG. 11. The results shown in FIG. 11 are alsosummarized in the column of “Activity of inhibiting the binding of 293cells” in FIG. 1. In the column of FIG. 1, the mark “◯” indicates thatthe cell adhesion was inhibited significantly by the antibody, and themark “X” indicates that the antibody exhibited no inhibiting activity.

<4-11-2> Inhibition of the Binding of CTGF with Rat Kidney-derivedBroblast Cell Line NRK-49F

The above-prepared various monoclonal antibodies (the finalconcentration: 20 μg/well, 6 μg/well or 2 μg/well) reactive to the humanCTGF were added into the wells of a microplate coated with therecombinant human CTGF (the coating concentration: 1 μg/well) preparedby the same procedure described in Example <4-3>.

The cells of rat kidney-derived fibroblast cell line NRK-49F (ATCCCRL1570) labeled with 2′,7′-bis(2-carboxyethyl)-5(6)-carboxyfluoresceintetraacetoxymethyl ester (BCECF; Molecular Probes Inc.) were added intoeach well (1×10⁴ cells/well). The plates were allowed to stand at 4° C.for 1 hour.

The floating cells were removed and then the cells adhering to theplates were solubilized by adding phosphate buffer (100 μl) containing1% NP-40 thereto. The release of BCECF into the culture supernatant wascaused by cytolysis. The intensity of fluorescence emitted by BCECF wasmeasured by the FLUOROSCAN II MICROPLATE FLUOROMETER (Flow LaboratoriesInc.).

Control assay was carried out in the same manner described above butwithout any antibodies added. Negative control assay was carried out byusing human monoclonal antibody against KLH (keyhole limpet hemocyanin;Pierce Chemical Co.) in the same manner described above.

The anti-KLH antibody was prepared by immunizing the above-describedhuman antibody-producing transgenic mice with KLH.

Another control assay was performed in the same manner described abovebut without any antibodies added and by using a microplate on which noCTGF was immobilized.

The results are shown in FIG. 12. The result is shown as a rate (%) ofbound cells, which is calculated based on the value of fluorescenceintensity determined.

The results shown in FIG. 12 are also summarized in the column of“Activity of Inhibiting the Binding of NRK Cells” in FIG. 2. In thecolumn of FIG. 2, the mark “◯” indicates that the cell adhesion wassignificantly inhibited by the antibody, and the mark “X” indicates thatthe antibody exhibited weak inhibiting activity or no activity.

<4-11-3> Inhibition of the Binding of CTGF with Various Cells

The above-prepared various monoclonal antibodies (the finalconcentration: 20 μg/well) reactive to human CTGF were added into thewells of microplate coated with the recombinant human CTGF (the coatingconcentration: 1 μg/well) prepared by the same procedure described inExample <4-3>.

After the plates were allowed to stand for 60 minutes and thesupernatants were removed from each plate, the cells (1×10⁴ cells/well)indicated below labeled with 2′,7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein tetraacetoxymethyl ester (BCECF; MolecularProbes Inc.) were added into each well. The plates were allowed to standat 4° C. for 1 hour. The following cells were used in this assay:

(1) human lung-derived fibroblast cell (NHLF2837; Clonetics);

(2) human osteosarcoma-derived cell line MG-63 (ATCC CRL1427); and

(3) rat kidney-derived fibroblast cell line NRK-49F (ATCC CRL1570).

The floating cells were removed and then the cells adhering to theplates were solubilized by adding phosphate buffer (100 μl) containing1% NP-40 thereto. The release of BCECF into the culture supernatant wascaused by cytolysis. The intensity of fluorescence emitted by BCECF wasmeasured in the FLUOROSCAN II MICROPLATE FLUOROMETER (Flow LaboratoriesInc.).

Control assay was carried out in the same manner described above butwithout any antibodies added. Negative control assay was carried out byusing human monoclonal antibody against KLH (keyhole limpet hemocyanin;Pierce Chemical Co.) in the same manner described above. The anti-KLHantibody was prepared by immunizing the above-described humanantibody-producing transgenic mice with KLH.

Another control assay was performed in the same manner described abovebut without any antibodies added and by using a microplate on which noCTGF was immobilized.

The results are shown in FIG. 13. The result is shown as a rate (%) ofbound cells, which is calculated based on the value of fluorescenceintensity determined.

<4-12> Crossreactivity to Rabbit Tissues

Arteriosclerotic lesions were obtained from the hyperlipidemia modelrabbit WHHL (Oriental Yeast Co., Ltd.) by a surgical operation.

Frozen sections were prepared from the arteriosclerotic lesionsaccording to a usual method.

Each section was stained by using a Vectastain Elite ABC kit (FunakoshiLtd.) according to the procedure described below.

After being fixed with acetone for 1-2 minutes and dried, the sectionswere moistened with a diluted serum (10 ml PBS/150 μl serum) for 30minutes. The sections were washed with PBS, and then primary antibodies(10 μg/ml or culture supernatant of hybridoma) were added. The primaryantibodies used are the above-mentioned anti-human CTGF monoclonalantibodies (clone: 8-86-2 and 8-149-3) derived from normal mouse;anti-mouse CTGF monoclonal antibody derived from normal rat (clone:13-51-2); and anti-human CTGF monoclonal antibodies derived from thehuman antibody-producing transgenic mouse (clone: A4.3, A11.1, A29.6,B29.6, B35.1, C26.11, and C114.4). The sections were allowed to standfor 40 minutes.

Subsequently, each section was washed with PBS, and then a solution ofbiotinylated secondary antibody (100 μl) was added. The sections wereallowed to stand for 30 minutes. Biotin-labeled horse anti-mouseimmunoglobulin antibody was used as the secondary antibody when theprimary antibody added was the anti-human CTGF monoclonal antibodyderived from normal mouse; biotin-labeled rabbit anti-rat immunoglobulinantibody was used as the secondary antibody when the primary antibodyadded was the anti-mouse CTGF monoclonal antibody derived from normalrat; biotin-labeled goat anti-human immunoglobulin antibody was used asthe secondary antibody when the primary antibody added was theanti-human CTGF monoclonal antibody derived from the humanantibody-producing transgenic mouse.

Each section was allowed to stand in a methanol solution containing 3%hydrogen peroxide for 10 minutes and then washed with PBS. Then 100 μlof an avidin-peroxidase solution (PBS (5 ml)/peroxidase-labeled avidinDH (100 μl)/biotinylated hydrogen peroxide H (100 μl ) was added to thesections. The sections were allowed to stand for 30 minutes.

After washing with PBS, a diaminobenzidine tetra hydrochloride solution(DAB)(Water (5 ml)/buffer solution (100 μl)/DAB solution (200μl)/hydrogen peroxide solution (100 μl )) was added to the sections. Thesections were allowed to stand for 2-10 minutes.

After washing with cold water for 5 minutes, the sections were subjectedby Giemsa staining method and mounted. Control staining was carried outin the same manner by using as a primary antibody a monoclonal antibody,which is non-reactive to CTGF and is identical in isotype to thecorresponding monoclonal antibody to be tested. The stained and mountedsections were observed under a microscope with magnifications of X100and X200. The results are shown in FIG. 14. The results shown in FIG. 14are also summarized in the column of “Reactivity to Tissues fromArteriosclerotic Lesions of WHHL rabbit” in FIG. 1. In the column ofFIG. 1, the mark “◯” indicates that the tissue was stained with theantibody, and the mark “X” indicates that the tissues was weakly stainedor not stained with the antibody.

Anti-human CTGF monoclonal antibody clones derived from the humanantibody-producing transgenic mouse, A4.3, A11.1, A29.6, C26.11 andC114.4, and a clone of anti-mouse CTGF monoclonal antibody derived fromnormal rat, 13-51-2, exhibited the reactivity to the tissues fromarteriosclerotic lesions of rabbit.

<4-13> Activity of Inhibiting Cell Proliferation by a Stimulus with CTGF

As shown above in Example <4-9>, CTGF induces proliferation of a varietyof cells (for example, fibroblast cells derived from various tissuessuch as the kidney and lung, a variety of tumor cells, and vascularendothelial cells).

In the present experiment, the inhibiting effect of the monoclonalantibodies of the present invention on the CTGF-stimulated cellproliferation was examined described below.

<4-13-1> Preparation of Cell Culture Medium Containing CTGF

Neonatal human dermal fibroblast cells (NHDF; Becton Dickinson) werecultured in a dish. The cells were washed twice with a fetal calf serum(FCS)-free DMEM medium and then further cultured with a fresh DMEMmedium containing human transforming growth factor-β (TGF-β; 1 ng/ml;R&D Systems) for 1 day.

The culture supernatant was recovered, and loaded onto to a heparincolumn (HITRAP; Pharmacia Biotech). The column was washed with 0.2MNaCl/PBS, and then the factor trapped in the column was eluted with 0.6MNaCl/PBS. The eluate was dialyzed with PBS and was used for the cellproliferation assay described below.

Because CTGF is a heparin-binding protein, CTGF can be partiallypurified by using a heparin column. The presence of CTGF in the sampleobtained above was confirmed according to a usual method by Westernblotting using the rabbit anti-human CTGF polyclonal antibody preparedin Example 1.

Control assay was carried out by using pre-immune serum prepared fromthe rabbit (the same rabbit described in Example 1) prior to theimmunization with human CTGF.

The results are shown in FIG. 24.

<4-13-2> Cell Proliferation by a Stimulus with Purified CTGF

The cells of rat kidney-derived fibroblast cell line NRK-49F (ATCCCRL-1570; 1×10⁴ cells/well) were placed in the wells of a 96-wellmicrotiter plate, and cultured for 1 day. The plate was washed twicewith a FCS-free DMEM medium. Then the cells were further cultured for 1day. Subsequently, the CTGF sample prepared above in <4-13-1> (diluted10, 30, 100 and 300 times with a DMEM medium) was added into each well,the culture was continued for 18 hour. [³H]-thymidine (3.7 kBq/well) wasadded into each well, and the culture was further continued for 6 hours.The cells were harvested for the measurement of [³H]-thymidine uptake bythe cells. The measurement was carried out in a liquid scintillationcounter (Beckman).

Positive control experiment was carried out with PDGF in the samemanner. Negative control experiment was performed in the same manner butwithout CTGF added.

The results are shown in FIG. 25.

<4-13-3> Activity of Inhibiting Cell Proliferation

The CTGF sample (diluted 20 times with a DMEM medium) prepared above in<4-13-1> was allowed to react for 30 minutes with the human anti-humanCTGF monoclonal antibodies of the present invention (the finalconcentration: 20 μg/ml, 2 μg/ml, or 0.2 μg/ml) that was prepared above.The same experiment described in <4-13-2> was carried out with themixture.

Positive control assay was carried out in the same manner but withoutany antibodies added. Negative control assay was performed in the samemanner in the absence of any of the CTGF samples and the antibodies.

The results are shown in FIGS. 15 and 16.

The same experiment described above was repeated several times. Theresults including those shown in FIGS. 15 and 16 are summarized in thecolumn “Activity of Inhibiting the proliferation of NRK Cells” of FIG.2. In the column of FIG. 2, the mark “◯” indicates that the cellproliferation was significantly inhibited by the antibody.

Thus, it was revealed that the human anti-human CTGF monoclonalantibodies of the present invention significantly suppressed orinhibited the proliferation of human fibroblast cells.

<4-14> Epitope Mapping

The experiment described below was performed to determine the sites(epitopes) in the structure of human CTGF responsible for the specificbinding with the human anti-human CTGF monoclonal antibodies of thepresent invention.

This experiment was carried out by the inventive sandwich ELISA 30 withtwo antibodies. The method of the sandwich ELISA of the presentinvention is described in detail in Example 5. Specifically, theexperiment was conducted according to the following steps:

(Step 1)

Antibody-immobilized microplates, on which each of the human 35anti-human CTGF monoclonal antibodies (0.3 μg/50 μl /well) listed inFIG. 2 was immobilized, were prepared in the same manner described laterin Example <5-1>.

(Step 2)

Labeled monoclonal antibodies were prepared in the same manner describedlater in Example <5-2>, by using the monoclonal antibodies, A, B, C andD, of the present invention, as follows:

[Antibody A]

Mouse monoclonal antibody 8-64-6 reactive to human CTGF (derived from ahybridoma identified by international deposit accession No. FERMBP-6209);

[Antibody B]

Human anti-human CTGF monoclonal antibody A11.1 (derived from ahybridoma identified by international deposit accession No. FERMBP-6535);

[Antibody C]

Human anti-human CTGF monoclonal antibody C26.11 (consisting of theheavy chain having an amino acid sequence comprising the amino acidsequence of SEQ ID NO: 8 as well as the light chain having an amino acidsequence comprising the amino acid sequence of SEQ ID NO: 18);

[Antibody D]

Human anti-human CTGF monoclonal antibody C59.1 (consisting of the heavychain having an amino acid sequence comprising the amino acid sequenceof SEQ ID NO: 10 as well as the light chain having an amino acidsequence comprising the amino acid sequence of SEQ ID NO: 20).

(Step 3)

ELISA was conducted in the same manner described later in Example <5-3>.Specifically, the purified recombinant human CTGF prepared in the aboveexample was added (15 ng/well) into the wells of eachantibody-immobilized microplate prepared in Step 1. After theantigen-antibody reaction was allowed to proceed, the respective labeledmonoclonal antibodies prepared in Step 2 were added (0.1 μl/50 μl/well)and reacted thereto. After the subsequent treatment conducted in thesame manner described later in Example <5-3>, the reaction was stoppedby adding a stop solution into each well. The intensity of fluorescenceemitted in each well was measured with the fluorometer at a wavelengthof 460 nm (excitation: 355 nm).

The value of fluorescence intensity obtained is expected to depend onthe positional relationship between the site (epitope) on the human CTGFbound with the monoclonal antibody immobilized on the microplate and thesite (epitope) on the human CTGF bound with the labeled monoclonalantibody. Accordingly, the results are predicted to be described belowin (1) to (3).

(1) When the site (epitope) on the human CTGF where the monoclonalantibody immobilized on the microplate binds, is identical to the site(epitope) on the human CTGF where the labeled monoclonal antibody binds,then the labeled antibody added later cannot react with theantigen-antibody complex consisting of the human CTGF and the monoclonalantibody immobilized on the microplate. Therefore, the value offluorescence intensity to be measured in Step 3 can be sufficiently near0 in this case.

(2) When the site (epitope) on the human CTGF where the monoclonalantibody immobilized on the microplate binds, is adjacent to the site(epitope) on the human CTGF where the labeled monoclonal antibody binds,then, because of some steric hindrance around the site, the labeledantibody added later becomes less reactive to the antigen-antibodycomplex consisting of the human CTGF and the monoclonal antibodyimmobilized on the microplate. Therefore, the value of fluorescenceintensity to be measured in Step 3 is expected to be low in this case.

(3) When the site (epitope) on the human CTGF where the monoclonalantibody immobilized on the microplate binds, is distant from the site(epitope) on the human CTGF where the labeled monoclonal antibody binds,then the labeled antibody added later can react with theantigen-antibody complex consisting of the human CTGF and the monoclonalantibody immobilized on the microplate. Therefore, the value offluorescence intensity to be measured in Step 3 can be significantlyhigh in this case.

The result of the experiment agreed with the above prediction. Theresult is shown in the column of “Epitope mapping” of FIG. 2.

The meaning of each alphabet used in the column of FIG. 2 is illustratedbellow.

“(A)” indicates the “Antibody A” used above as the labeled antibody;

“(B)” indicates the “Antibody B” used above as the labeled antibody;

5 “(C)” indicates the “Antibody C” used above as the labeled antibody;

“(D)” indicates the “Antibody D” used above as the labeled antibody;

“A” indicates an epitope identical or almost identical to the epitope of“Antibody A”;

“B” indicates an epitope identical or almost identical to the epitope of“Antibody B”;

“C” indicates an epitope identical or almost identical to the epitope of“Antibody C”;

“D” indicates an epitope identical or almost identical to the epitope of“Antibody D”;

“-” indicates an epitope which is different from any of the epitopes of“Antibody A,” “Antibody B,” “Antibody C” and “Antibody D”;

“B/C” indicates an epitope identical or almost identical to the epitopeof “Antibody B” and/or the epitope of “Antibody C”;

“A-/B” indicates an epitope close to the epitope of “Antibody A” inposition and identical or almost identical to the epitope of “AntibodyB.”

Others are indicated in the same manner as indicated above.

<4-15> Therapeutic Effect on Kidney Diseases and Fibrotic Diseases inTissues

The therapeutic effect of the human anti-human CTGF monoclonalantibodies of the present invention on various diseases was studied byusing a mouse model of the diseases.

The mouse model used in this study is a disease model exhibiting a partof the pathologic features or a part of clinical findings as observed inany of the diseases and morbid conditions indicated below.

The therapeutic effect found in the present study should be consideredto represent all of the therapeutic effects on the diseases or morbidconditions indicated below:

kidney diseases (renal failure, nephritis, kidney fibrosis, etc.),various fibrotic diseases in tissues (kidney fibrosis, pulmonaryfibrosis, hepatic fibrosis, tissue fibrosis of skin, etc., fibrosis ofsynovial tissue associated with rheumatoid arthritis and fibroticdiseases associated with various cancers), skin diseases (scleroderma,psoriasisa, atopic dermatitis, etc.), liver diseases (cirrhosis, hepaticfibrosis, hepatitis, etc.), lung diseases (pulmonary fibrosis,pneumonia, etc.) and rheumatoid arthritis and arteriosclerosis, etc.

<4-15-1> Preparation of a Disease Model Mouse

The left side abdomen of each B6C3F1 mouse (male, 7-week old, 6individuals per each group, SLC) was opened by a surgical operationunder anesthesia with pentobarbital (50 mg/kg). The ureter extendingfrom the left kidney was ligated with sutures at two sites and then cutbetween the two ligated sites (UUO, unilateral ureteral obstruction).After this treatment, the opened abdominal part was sutured. Thisoperation results in the loss of the most important function in the leftkidney-the normal renal filtering function of body fluids such as blood.A variety of pathological manifestations as seen in various kidneydiseases are observed in the operated kidney.

<4-15-2> Therapeutic Effect of Anti-CTGF Monoclonal Antibody

The human anti-human CTGF monoclonal antibody, M84 or M32 (prepared inthe previous Example) was dissolved in a phosphate buffer and given tothe above-prepared model mice by intraperitoneal injection (5 mg/kg).The administration of the antibody was carried out, for the first time,immediately after the operation, and then every third day, four times intotal. After the final administration (14 days after the operation), theleft kidney was removed from each mouse by a surgical operation. Theextracted kidneys were delipidated and dehydrated with acetone, and theproteins were hydrolyzed by using 6N hydrochloric acid. Subsequently,the samples were dried, while being kept warm and blown with nitrogengas, and dissolved in purified water to serve as the assay samples. Thecontents of hydroxyproline (OH-proline) in the samples from kidneytissues were determined according to a previously reported method(Analytical Biochemistry, Vol. 55, p. 288-291, 1973; Kidney Lnt., Vol.54, No. 1, p. 99-109, 1998).

The increased hydroxyproline level in the kidney is an index of theonset of nephritis and kidney fibrosis resulting from renal failure.Accordingly, the decrease in the hydroxyproline concentration indicatesthat the monoclonal antibodies are useful for the treatment of kidneydiseases. Control experiments were carried out as follows in the samemanner described above:

(1) Phosphate buffer alone (without any antibodies) was givenintraperitoneally to mice treated with the above-mentioned UUO (ureterligation treatment) in the same manner described above.

(2) The abdomen was opened and then sutured without performing UUO innormal mice.

(3) No surgical operation with the UUO treatment was performed in normalmice.

(4) Mice were fed with food in which Pirfenidone (Kidney Int. Vol. 54,No. 1, p. 99-109, 1998) was mixed, instead of the antibody treatmentdescribed above (positive control experiment). Pirfenidone is a drug forthe treatment of fibrotic diseases such as kidney fibrosis and is beingunder clinical trial.

The results are shown in FIG. 17.

These results illustrated that the monoclonal antibodies of the presentinvention had significant suppressing effects and therapeutic effects onthe kidney diseases and tissue fibrotic diseases.

Surprisingly, the efficacy of the monoclonal antibodies of the presentinvention is the same as that of the drug used in a high dose as apositive control (for example, the amount of the drug given to micecorresponds to about 100 g, in total, if the drug is administered fourtimes to a patient with 50 kg of body weight).

<4-16> Determination and Analysis of Gene Sequence and Amino AcidSequence of Human Anti-human CTGF Monoclonal Antibody

Nucleotide sequencing was carried out as described below to determinethe cDNA sequence encoding the variable region of the heavy chain ofeach human monoclonal antibody against human CTGF prepared in the aboveexamples as well as the cDNA sequence encoding the viable region andconstant regions of the light chain of the antibody. The structuralfeatures of the genes were also analyzed. The procedures of sequencinganalysis used in this example is schematically illustrated in FIG. 18.

After culturing, the hybridomas (clone: A29, C26, C59, C114, and M295;about 5×10⁷ cells), which were prepared in the previous example,producing the human anti-human CTGF monoclonal antibodies, werecentrifuged and the resulting precipitates were recovered. The cellswere stored at −80° C. for the later polyA+RNA extraction.

PolyA⁺ RNA was extracted and purified from each hybridoma using acommercial product, FASTRACK 2.0 KIT (Invitrogen Co.), as follows. Eachof the above frozen cell samples were lysed in a lysis buffer andsolubilized by homogenizing with POLYTRON. After incubating at 45° C.,the solubilized materials were mixed with Oligo (dT) cellulose, and themixtures were shaken gently for about 1 hour. Subsequently, the Oligo(dT) cellulose was washed, and then polyA⁺ RNAs were eluted with anelution buffer. Eluted PolyA⁺ RNAs were precipitated with ethanol, andthen dissolved in 20 μl of Tris-EDTA buffer. The concentrations ofobtained polyA⁺ RNAs were determined by measuring absorbance at a wavelength of 260nm.

Complementary DNAs were prepared by using the obtained polyA+RNAs astemplates by RACE-PCR with a commercially available product, MARATHONcDNA AMPLIFICATION KIT (CLONTECH), according to a usual procedure (“PCRMethod for Gene Amplification: Basic Techniques and Recent Advancement”Kyoritsu Shuppan Co., Ltd., p.13-15, 1992). Specifically, thefirst-strand cDNA synthesis was performed using the polyA⁺ RNA (1-5 μg)purified from each of the hybridomas as a template, and then the secondstrand was prepared from the first strand. The cDNAs were extracted oncewith phenol/chloroform/isoamyl alcohol and then treated once withchloroform. The cDNAs were precipitated with ethanol. An adaptor DNA(SEQ ID NO: 25) was ligated to the cDNAs. The resultant DNA productswere diluted 250 times. The respective cDNAs encoding the heavy chainand the light chain of the antibody were prepared by using the dilutesas templates by PCR in a usual manner. The primer of SEQ ID NO: 26 wasused in the PCR for the antibody heavy chain. The primer of SEQ ID NO:27 was used in the PCR for the antibody light chain.

Each PCR product was fractionated by agarose-gel electrophoresis, andthe DNAs of interest were recovered therefrom. The nucleotide sequencesof the respective cDNAs obtained were determined by using a commerciallyavailable reagent, DYE TERMINATOR CYCLE SEQUENCING FS KIT (PE-AppliedBiosystems) and a PRISM377 DNA Sequencer (PE-Applied Biosystems).Sequencing Primers used in the sequence determination were the same asthose used in the above PCR amplification. Based on the sequencesobtained, appropriate sequencing primers were designed and prepared forfurther sequencing analysis.

Sequence listing attached hereto contains the CDNA sequence encoding thevariable region of the heavy chain of each human monoclonal antibodyagainst human CTGF produced by the above-mentioned hybridomas; the cDNAsequence encoding the variable region of the light chain of each of theantibodies; and the deduced amino acid sequences thereof.

<Clone A29>

(Variable Region of the Heavy Chain)

DNA sequence: SEQ ID NO: 5 (signal sequence: nucleotides 1-57, V region:nucleotides 58-363)

Amino acid sequence: SEQ ID NO: 6 (signal sequence: amino acids 1-19,variable region: comprising amino acids 21-120)

(Variable Region of the Light Chain)

DNA sequence: SEQ ID NO: 15 (signal sequence: nucleotides 1-60, Vregion: nucleotides 61-365)

Amino acid sequence: SEQ ID NO: 16 (signal sequence: amino acids 1-20,variable region: comprising amino acids 21-120)

<Clone C26>

(Variable Region of the Heavy Chain)

DNA sequence: SEQ ID NO: 7 (signal sequence: nucleotides 1-30 57, Vregion: nucleotides 58-357)

Amino acid sequence: SEQ ID NO: 8 (signal sequence: amino acids 1-19,variable region: comprising amino acids 21-118)

(Variable Region of the Light Chain)

DNA sequence: SEQ ID NO: 17 (signal sequence: nucleotides 1-60, Vregion: nucleotides 61-364)

Amino acid sequence: SEQ ID NO: 18 (signal sequence: amino acids 1-20,variable region: comprising amino acids 21-121)

<Clone C59>

(Variable Region of the Heavy Chain)

DNA sequence: SEQ ID NO: 9 (signal sequence: nucleotides 1-57, V region:nucleotides 58-350)

Amino acid sequence: SEQ ID NO: 10 (signal sequence: amino acids 1-19,variable region: comprising amino acids 21-116)

(Variable Region of the Light Chain)

DNA sequence: SEQ ID NO: 19 (signal sequence: nucleotides 1-66, Vregion: nucleotides 67-353)

Amino acid sequence: SEQ ID NO: 20 (signal sequence: amino acids 1-22,variable region: comprising amino acids 23-117)

<Clone C114>

(Variable Region of the Heavy Chain)

DNA sequence: SEQ ID NO: 11 (signal sequence: nucleotides 1-57, Vregion: nucleotides 58-350)

Amino acid sequence: SEQ ID NO: 12 (signal sequence: amino acids 1-19,variable region: comprising a segment of amino acids 21-116)

(Variable Region of the Light Chain)

DNA sequence: SEQ ID NO: 21 (signal sequence: comprising nucleotides1-47, V region: nucleotides 48-335)

Amino acid sequence: SEQ ID NO: 22 (signal sequence: comprising aminoacids 1-16, variable region: comprising amino acids 17-111)

<Clone M295>

(Variable Region of the Heavy Chain)

DNA sequence: SEQ ID NO: 13 (signal sequence: nucleotides 1-58, Vregion: nucleotides 59-353)

Amino acid sequence: SEQ ID NO: 14 (signal sequence: amino acids 1-19,variable region: comprising amino acids 21-117)

(Variable Region of the Light Chain)

DNA sequence: SEQ ID NO: 23 (signal sequence: nucleotides 1-66, Vregion: nucleotides 67-356)

Amino acid sequence: SEQ ID NO: 24 (signal sequence: amino acids 1-22,variable region: comprising amino acids 23-118)

By using a gene sequence-analyzing computer software, a library ofvariable region genes of human immunoglobulin, V BASE SEQUENCE,constructed by Tomlinson et al. (Immunol. Today, Vol. 16, No. 5, p.237-242, 1995) was searched for the respective DNA sequences determined.

The result showed that the respective V region genes of the heavy andlight chains of the above-mentioned human monoclonal antibodies arecomposed of the segments indicated below.

<Gene for Heavy-chain V Region>

Clone A29: DP-38

Clone C26: DP-75

Clone C59: DP-5

Clone C114: DP-5

Clone M295: DP-65

<Gene for Light-chain V Region>

Clone A29: DPK24

Clone C26: DPK12

Clone C59: DPK1

Clone C114: DPK1

Clone M295: DPK9

It is assumed that N-additions are located between the V region and thedownstream D region as well as between the D region and the furtherdownstream J region in the cDNA sequences encoding the heavy chains ofthe above human monoclonal antibodies.

EXAMPLE 5 Establishment of a Sandwich ELISA System for Assaying HumanCTGF and Mouse CTGF

<5-1> Preparation of a Antibody-immobilized Microplate

In this example, the monoclonal antibody 8-64-6 (derived from ahybridoma identified by international deposit accession No. FERMBP-6209), which was derived from normal mouse and prepared in theabove-described manner, was used as a monoclonal antibody which isimmobilized on a microplate. This monoclonal antibody is highly reactiveto human CTGF and crossreactive to mouse CTGF.

The monoclonal antibody 8-64-6 was diluted with phosphate buffer andadded at a concentration of 1 μg/50 μl/well into each well of a 96-wellELISA microplate (Corning Costar Co.). The plate was incubated at roomtemperature for 1 hour for adsorbing the antibody onto the plate.

Subsequently, the plate was washed with a phosphate buffer and then aphosphate buffer (200 μl/well) containing 3% bovine serum albumin (BSA)was added into each well. The plate was incubated at room temperaturefor 2 hours for the blocking of antibody-free sites on the microplate.The plate was washed three times with phosphate buffer.

<5-2> Preparation of a Labeled Monoclonal Antibody

In this example, the monoclonal antibody 8-86-2 (derived from ahybridoma identified by international deposit accession No. FERMBP-6208), which was derived from normal mouse and prepared in theabove-described manner, was used as a monoclonal antibody for thelabeling. This monoclonal antibody is highly reactive to human CTGF,mouse CTGF, and rat CTGFs.

One milliliter of the monoclonal antibody 8-86-2 (20 mg/ml) was dialyzedwith 0.1M NaHCO3 (pH8.2-8.3) solution (at 4° C. for 24 hours). Then 100μl of NHS-biotin (2 mg/ml; Pierce Chemical Co.) was added thereto andstirred vigorously. After the reaction was continued at room temperaturefor 30 minutes, the mixture was dialyzed with phosphate buffer (at 4° C.for 24 hours).

<5-3> Establishment of a Assay Method Using Sandwich ELISA

The sandwich ELISA system for assaying human CTGF and mouse CTGF, whichwas established in the present invention, is as follows.

Samples (50 μl/well) to be assayed were added into each well of theantibody-immobilized microplate prepared in Example <5-1> and incubatedat room temperature for 1 hour. The microplate was washed three timeswith phosphate buffer containing 0.1% Tween 20. The biotin-labeledmonoclonal antibody prepared in Example <5-2> was diluted with aphosphate buffer containing 1% BSA and 0.1% Tween 20 and added (0.3μl/50 μl/well) into the respective wells. The plate was incubated atroom temperature for 1 hour.

The microplate was washed three times with phosphate buffer containing0.1% Tween 20. A solution of streptavidin-β-galactosidase (50/11;Gibco-BRL), diluted 1000 times with a solution (pH7.0) containing 20 mMHEPES, 0.5M NaCl and BSA (1 mg/ml), was added into each well. The platewas incubated at room temperature for 30 minutes.

The microplate was washed three times with phosphate buffer containing0.1% Tween 20. A solution of 1% 4-Methyl-umbelliferyl-β-D-galactoside(50 μl; Sigma) in a phosphatebuffer (10 mM, pH7.0, containing Na and Kions) containing 100 mM NaCl, 1 mM MgCl₂ and 1 mg/ml BSA, was added intoeach well. The plate was incubated at room temperature for 10 minutes.

1M Na₂CO₃ (100 μl) was added to each well to stop the reaction. Thefluorescence intensity was measured by the FLUOROSCAN II MICROPLATEFLUOROMETER (Flow Laboratories Inc.) at a wavelength of 460nm(excitation wavelength: 355 nm). The amount of human CTGF or mouse CTGFin the sample were determined by using the calibration curves asprepared in the following example.

<5-4> Preparation of the Calibration Curve

The calibration curve was prepared by using the sandwich ELSAestablished in Example <5-3>, in which the labeled CTGF standard usedwas the affinity-purified recombinant human CTGF or recombinant mouseCTGF, which were prepared in Example <4-8>. The result is shown in FIG.19.

The calibration curve for human CTGF was obtained with a significantdifference even within an extremely low concentration range of 3 ng/mlto 1000 ng/ml. The calibration curve for mouse CTGF was also obtainedwith a significant difference for a concentration range of 30 ng/ml to1000 ng/ml. However, rat CTGF was not measurable in the sandwich ELISAsystem established in Example <5-3>.

EXAMPLE 6 Establishment of a Sandwich ELISA System for Assaying MouseCTGF and Rat CTGF

<6-1> Preparation of Antibody-immobilized Microplate

In this example, the monoclonal antibody 13-51-2, which was derived fromnormal rat and prepared in the above-described manner, was used as amonoclonal antibody which is immobilized on a microplate. Thismonoclonal antibody is highly reactive to mouse CTGF and crossreactiveto rat CTGF.

The monoclonal antibody 13-51-2 was diluted with phosphate buffer andadded at a concentration of 1 μg/50 μl/well into each well of a 96-wellELISA microplate (Corning Costar Co.). The plate was incubated at roomtemperature for 1 hour for adsorbing the antibody onto the plate.

Subsequently, the plate was washed with a phosphate buffer and then aphosphate buffer (200 μl /well) containing 3% Bovine serum albumin (BSA)was added into each well. The plate was incubated at room temperaturefor 2 hours for the blocking of antibody-free sites on the microplate.The plate was washed three times with phosphate buffer.

<6-2> Preparation of a Labeled Monoclonal Antibody

In this example, labeled monoclonal antibody was the biotin-labeledmonoclonal antibody prepared in Example <5-2>.

Specifically, the labeled monoclonal antibody was prepared by labeling,with biotin, the antibody 8-86-2 (derived from a hybridoma identified byinternational deposit accession No. FERM BP-6208) highly reactive to allof human CTGF, mouse CTGF, and rat CTGF.

<6-3> Establishment of a Assay Method Using Sandwich ELISA

The sandwich ELISA system for assaying mouse CTGF and rat CTGF, whichwas established in the present invention, is as follows. Samples (50μl/well) to be assayed were added into each well of theantibody-immobilized microplate prepared in Example <6-1> and incubatedat room temperature for 1 hour. The microplate was washed three timeswith phosphate buffer containing 0.1% Tween 20. The biotin-labeledmonoclonal antibody prepared in Example <6-2> was diluted with aphosphate buffer containing 1% BSA and 0.1% Tween 20 and added (0.3μl/50 μl/well) into the respective wells. The plate was incubated atroom temperature for 1 hour.

The microplate was washed three times with phosphate buffer containing0.1% Tween 20. A solution of streptavidin-β-galactosidase (50 μl;Gibco-BRL), diluted 1000 times with a solution (pH7.0) containing 20 mMHEPES, 0.5M NaCl and BSA (1 mg/ml), was added into each well. The platewas incubated at room temperature for 30 minutes.

The microplate was washed three times with phosphate buffer containing0.1% Tween 20. A solution of 1% 4-Methyl-umbelliferyl-β-D-galactoside(50 μl; Sigma) in a phosphate buffer (10 mM, pH7.0, containing Na and Kions) containing 100 mM NaCl, 1 mM MgCl₂ and 1 mg/ml BSA, was added intoeach well. The plate was incubated at room temperature for 10 minutes.

1M Na₂CO₃ (100 μl) was added to each well to stop the reaction. Thefluorescence intensity was measured by the FLUOROSCAN II MICROPLATEFLUOROMETER (Flow Laboratories Inc.) at a wavelength of 460 nm(excitation wavelength: 355 nm). The amount of mouse CTGF or rat CTGF inthe sample were determined by using the calibration curve as prepared inthe following example.

<6-4> Preparation of the Calibration Curve

The calibration curve was prepared by using the sandwich ELSAestablished in Example <6-3> in which the labeled CTGF standard used wasthe affinity-purified recombinant mouse CTGF or recombinant rat CTGF,which were prepared in Example <4-8>. The result is shown in FIG. 20.

The calibration curve for mouse CTGF was obtained with a significantdifference even for an extremely low concentration range of 1 ng/ml to1000 ng/ml. The calibration curve for rat CTGF was also obtained with asignificant difference for a concentration range of 10 ng/ml to 1000ng/ml. However, human CTGF was not measurable in the sandwich ELISAsystem established in Example <6-3>.

EXAMPLE 7 Assay of CTGF in the Sera from Patients Affected with VariousDiseases

CTGF in the sera from patients affected with various diseases wasassayed by the sandwich ELISA established in Example <5-3>.

<7-1> Biliary Atresia, Rheumatic Vasculitis, Malignant RheumatoidArthritis, Psoriasis, and Atopic Dermatitis

Human sera used in this experiment were collected from normal healthypersons (33 samples), patients affected with biliary atresia andsubmitted to a surgical operation (post-operative sample; <Group 1>patients with normal clinical findings (17 samples); <Group 2> patientswith progressing symptoms (14 samples); <Group 3> patients with severesymptoms in need of liver transplantation (8 samples)), patients withrheumatic vasculitis (10 samples), patient with malignant rheumatoidarthritis (MRA)(17 samples), patients with psoriasis (24 samples) andpatients with atopic dermatitis (34 samples).

The results are shown in FIG. 21 (biliary atresia) and FIG. 22(rheumatic vasculitis, malignant rheumatoid arthritis, psoriasis, andatopic dermatitis).

It was evidenced that, among patients affected with biliary atresia,CTGF was significantly expressed in Group 2 patients (with symptoms atprogressive stage). In addition, as compared with normal healthypersons, patients affected with rheumatic vasculitis or malignantrheumatoid arthritis exhibited significantly higher expression of CTGF.

<7-2> Rheumatoid Arthritis and Osteoarthritis

Samples used in this experiment were synovial fluids collected frompatients affected with rheumatoid arthritis (RA; 36 patients) andpatients with osteoarthritis (OA; 19 patients). The result is shown inFIG. 23.

It was found that the synovial fluid CTGF levels of patients withrheumatoid arthritis were significantly higher than those of patientswith osteoarthritis.

Based on this result, it can be stated that the expression of CTGF inpatients with various diseases as well as normal healthy persons can behighly sensitively quantified by the assay system of the presentinvention and that the system can be utilized as a clinical diagnosisfor accurate clinical judgment of the degree of illness.

EXAMPLE 8 Preparation of Antibody Fragments F(ab′ )₂ and Fab

The antibody fragments F(ab′ )₂ and Fab derived from various monoclonalantibodies prepared above, are prepared as follows.

A sodium acetate buffer (20 mM; pH3.5) containing monoclonal antibody (5mg/ml) is incubated at 37 ° C. for 30 minutes. Insolubilized pepsin (1ml; Pierce Chemical Co.) is added thereto. The mixture is then incubatedat 37° C. for 12 hours while being shaken on a rotator. The reactionsolution is centrifuged (3000 rpm, for 10 minutes) and the resultingsupernatant is recovered.

Protein A-affinity chromatography is performed by using a Protein Acolumn kit (Amersham) according to the supplier's protocol, as follows.A binding buffer is added to the precipitate obtained by centrifugation.The solution is centrifuged (3000 rpm, for 10 minutes) again, and thenthe resulting supernatant is recovered. The first and secondsupernatants obtained are combined together and an equal volume of thebinding buffer is added thereto. The mixture is adjusted to pH 8.9 byadding 1N sodium hydroxide thereto. The mixed solution is loaded ontothe Protein A column equilibrated with the binding buffer. Then thecolumn is washed twice with the binding buffer (5 ml) to elute andcollect the fraction of interest. The fraction is dialyzed with 5 mMphosphate buffer (2 L, pH6.8) (at 4° C., for 24 hours).

Further purification is performed by high performance liquidchromatography (HPLC) using hydroxyapatite column (BioRad). The samplesolution after dialysis is loaded onto the hydroxyapatite column. 5 mMphosphate buffer is allowed to flow through the column for 15 minutes,the antibody fragments are eluted with a linear gradient of 5 mM-0.4Mphosphate buffer. The eluate is collected on a fraction collector andthe absorbance is monitored at a wavelength of 280 nm for the recoveryof F(ab′)₂-containing fractions. The fractions collected are dialyzedwith 2L of phosphate buffer (at 4° C., for 24 hours). The purified F(ab′)₂ of monoclonal antibody is thus obtained.

EXAMPLE 9 Preparation of Human-CTGF-expressing Transgenic Mouse

The human CTGF-encoding cDNA obtained in Example 2 was blunted by usinga DNA blunting kit (Takara Shuzo Co.) and inserted into an expressionvector, PCAGGS (Gene, Vol. 108, p. 193-200, 1991), containing chickenβ-actin promoter to obtain the plasmid phCTGF. Human kidney-derivedfibroblast cell line 293-T (ATCC CRL1573) was transformed byelectroporation with phCTGF. It was verified that the transformantexpressed and secreted human CTGF into the culture supernatant by thesandwich ELISA established in Example 5.

The plasmid phCTGF was linearized by the treatment with restrictionenzyme for the subsequent preparation of transgenic mouse.

White ICR females with a copulatory plug were selected as foster mothersthat were obtained by mating white ICR female mouse(Japan SLC) with avasectomized white ICR male mouse(Japan SLC). Donor female mice givingfertilized eggs to be used for human CTGF gene transfer were prepared bymating a female BDF-1 mouse (Japan SLC), which were superovulated by theadministration of PEAMEX (5 units; Sankyo Zohki Co.) and PREGNYL (5units; Organon Co.), with a BDF-1 male mouse (Japan SLC). After themating, the oviduct was removed from the BDF-1 mouse (female), and washyaluronidase-treated to obtain only fertilized eggs. The eggs werestored in a medium.

The human CTGF gene was introduced into the fertilized eggs by using amanipulator under a microscope according to the usual method. Fertilizedeggs were held-in-place with a holding needle. The above-mentionedlinear human CTGF gene, which was dissolved in Tris-EDTA buffer, wasmicroinjected into the male pronucleus of the egg with a DNA injectionneedle at 37° C.

After the gene transfer, only fertilized eggs with a normal appearancewere selected. The eggs, to which the human CTGF was introduced, weretransferred into the fimbria of the oviduct in the ovary of the mouse(white ICR mouse) used as a foster mother.

Genomic DNA was extracted form the tails of the resulting offspring(chimeric mice) born from the foster mother. The presence of thetransferred human CTGF gene in mouse genome was confirmed by PCR. Inaddition, it was confirmed that human CTGF was expressed and secretedinto blood serum of the mouse, by the sandwich ELISA established byExample 5. Then the chimeric mice were mated with normal mice to prepareheterozygous transgenic mice expressing human CTGF at high levels. Theheterozygous mice were mated to each other to prepare homozygoustransgenic mice.

EXAMPLE 11 Preparation of Rat CTGF

<11-1> cDNA Cloning

(1) Preparation of Rat cDNA Library and Probe

Cells of rat kidney-derived fibroblast strain NRK-49F (ATCC CRL-1570;about 1×10⁶ cells/ml) were centrifuged (at 4° C., 2,000×g, for 5minutes). The cells precipitated were suspended in ISOGEN (Nippon Gene)and then chloroform was added thereto. After the mixture was shaken, theupper layer was recovered. Isopropanol was added to the obtained uppersolution. The mixture was allowed to stand at room temperature for 10minutes and centrifuged (at 4° C., 12,000×g, for 10 minutes) for RNAprecipitation. After washing with ethanol, the precipitated RNA wasdissolved in TE buffer. Poly(A)⁺ RNA was purified from the total RNAusing an mRNA Purification Kit (Pharmacia).

Complementary DNA synthesis was performed by using the poly (A)+RNA (5μg) as a template and a SUPERSCRIPT 1 SYSTEM FOR cDNA SYNTHESIS KIT(GIBCO-BRL). An oligo dT primer with NotI site (GIBCO-BRL) was used forimproved screening efficiency. After linking to a SalI adaptor, cDNA wasdigested with NotI to give unidirectional CDNA. The heterogeneous cDNAwas fractionated by using a cDNA size fractionation column (GIBCO-BRL).

The nucleotide sequences of the human and mouse cDNAs obtained inExample 2 were compared to each other. A pair of 5′ (SEQ ID NO: 3) and3′ (SEQ ID NO: 4) primers were designed and synthesized based on ahighly homologous region shared by the human and mouse CTGF cDNAs.

PCR (polymerase chain reaction) amplification was performed by using theCDNA library prepared above as a template and by using theabove-mentioned primers and Ex Taq DNA polymerase (Takara Shuzo Co.).The reaction was carried out on a DNA THERMAL CYCLER (Perkin ElmerCetus). The final concentration of each primer was 0.4 μM and that ofMg²⁺ was 1.5 mM. The cycling profile was 35 cycles of the followingcycle: denaturing at 94° C. for 1 minute, annealing at 55° C. for 1minute and extension at 72° C. for 1 minute. After electrophoresed on anagarose gel, the amplified DNA was purified by using a QUIAEX DNAextraction kit (QUIAGEN).

The recovered DNA fragment was ligated with a vector pCRII (InvitrogenCo.) by using the TA CLONING KIT (Invitrogen Co.). Nucleotide sequenceof the resulting cDNA was sequenced on an A.L.F. DNA SEQUENCER(Pharmacia) by using an AUTO READ SEQUENCING KIT (Pharmacia) accordingto the dideoxy method. The nucleotide sequence of the cDNA fragment wascompared with those of human and mouse CTGF cDNAs obtained in Examples 2and 3. It was confirmed that the CDNA fragment contained the codingregion for rat homologue (rat CTGF) corresponding to the human and mouseCTGF gene.

A probe for plaque hybridization was prepared by labeling the cDNA(about 0.8 kb) with FITC by using an ECL RANDOM PRIME LABELING KIT(Amersham).

(2) Ligation of CDNA to Vector and Packaging

The cDNA obtained above in (1) was ligated to a vector 1zipLox NotI-SalIarm (GIBCO-BRL). The ligation reaction was performed by using a DNAligation Kit (Takara Shuzo Co.). The ligated DNA was packaged in vitroby using a GIGA PACK II GOLD (Stratagene). A cDNA library comprisingrecombinant phage-containing plaques was prepared with the obtainedphage particles and E. coli host Y1090 strain (GIBCO-BRL).

(3) Screening of a CDNA Library

According to the plaque hybridization method described in “MolecularCloning: A Laboratory Manual (Maniatis et al., Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y.)”, screening of the CDNA libraryprepared above in (2) was carried out by using RAPID HYBRIDIZATIONBUFFER (Amersham), as follows.

After the cDNA (1×10⁴ plaques) prepared above in (2) was seeded on agarplates, filter replicas were prepared with HYBOND-N-NYLON MEMBRANES(Amersham). Plaque hybridization was performed in the RAPIDHYBRIDIZATION BUFFER (Amersham) by using the FITC-labeled probe preparedabove in (1) and the replicas. The primary screening and the secondaryscreening yielded 13 positive clones. Each clone obtained bysingle-plaque isolation was treated by in vivo excision method accordingto the manual provided by the supplier GIBCO-BRL. Thirteen clones wereobtained as plasmid DNAs.

(4) Determination of Nucleotide Sequence

Nucleotide sequence of the 13 cDNA cones were determined on an A.L.F.DNA SEQUENCER (Pharmacia) by using an AUTO READ SEQUENCING KIT(Pharmacia) according to the dideoxy method. All 13 clones shared thesame nucleotide sequence. Comparison of the cDNA sequences with those ofhuman and mouse CTGFs revealed that an obtained clone, r311, contained afull-length cDNA encoding rat CTGF. The full-length CDNA sequence(comprising the nucleotide sequence of the 5′ end and the 3′ endthereof) of rat CTGF is shown in SEQ ID NO: 1, and the deduced aminoacid sequence thereof is shown in SEQ ID NO: 2.

<11-2> Preparation of Recombinant Rat CTGF

The clone r311 prepared in Example <11-1>, which contained a cDNAencoding rat CTGF, was digested with SalI and DraI, to obtain thefragment of rat CTGF-encoding cDNA. The DNA fragment was inserted into aplasmid pcDNA3.1 (−) (Invitrogen Co.) to prepare an expression vector.Human epithelioid cell line Hela cell (ATCC CCL-2) was transformed withthe vector by electroporation. GENETICIN-resistant transformants wereselected by culturing the transformed cells in an RPMI1640 mediumcontaining GENETICIN (0.8 mg/ml; GIBCO-BRL) and 10% fetal calf serum forabout two weeks. The selected transformants were cultured in aserum-free medium ASF104 (Ajinomoto Co. Inc.) for the expression of therecombinant rat CTGF. The expression of rat CTGF was confirmed byWestern blotting using the monoclonal antibody, which was prepared abovein Example 4, having the crossreactivity to rat CTGF.

The culture supernatant was recovered and treated by ammonium sulfateprecipitation method. The precipitated proteins were fractionated byheparin-column chromatography. The column was washed with 0.3M NaCl/PBS,and then the protein fraction of interest was eluted with 0.5M NaCl/PBS.Thus, a fraction with partially purified rat CTGF was obtained.

Industrial Applicability

The present invention provides previously unavailable various monoclonalantibodies derived from a variety of mammals. The antibodies arereactive to CTGFs from a variety of mammals such humans, mice, rats andrabbits, and are different from one another in respect to the propertiessuch as antigenic specificity, affinity for the antigen, neutralizingactivity, and crossreactivity. Particularly, the present invention leadsthe way in the world in providing various human monoclonal antibodiesagainst human CTGF by using, as an immune animal, the transgenic mouseprepared to produce human antibodies by recombinant technology.

Among the monoclonal antibodies of the present invention, the anti-humanCTGF monoclonal antibody and the pharmaceutical composition thereof,suppress and inhibit the onset and advancement of various diseases whichare believed to be caused by CTGF; such diseases include, for example,kidney diseases (kidney fibrosis, nephritis, and renal failure, etc.),lung diseases (for example, pulmonary fibrosis and pneumonia), liverdiseases (for example, hepatic fibrosis, cirrhosis, and hepatitis), skindiseases (for example, injuries, scleroderma, psoriasis, and keloid),arthritis (for example, rheumatoid arthritis and osteoarthritis), andvascular diseases (for example, rheumatic vasculitis), tissue fibrosisdeveloped as a complication in various cancers, and arteriosclerosis(specifically, tissue fibrosis which occurs as a complication). Theanti-human CTGF monoclonal antibody and the pharmaceutical compositionthereof are thus useful as pharmaceuticals for treating or preventingthese diseases.

The utility value of the antibody as a pharmaceutical is dramaticallyelevated, because the human monoclonal antibodies and the pharmaceuticalcomposition thereof are nonantigetic in humans, the antigenicity being amajor therapeutic problem (side effect) in the treatment with antibodypharmaceuticals comprising antibodies derived from non-human mammalssuch as mice.

By using immunoassay with the monoclonal antibodies of the presentinvention, it is possible to provide various immunoassay systems(methods and kits) for conveniently and highly sensitively assayingintact CTGF in the body fluids (such as serum) from mammals (human,mouse, rat and rabbit). It is also possible to easily purify CTGFs ofhigh purity from various mammals by using affinity column chromatographywith an insoluble carrier on which the monoclonal antibody isimmobilized.

Moreover, the inventive non-human transgenic mammal (transgenic mouse,etc.) expressing human CTGF is extremely useful as a tool for screeningcandidate pharmaceutical agents (low molecular weight compounds,antibodies, antisense nucleotides, and polypeptides except human CTGF)having the activity of regulating human CTGF functions (inhibition,suppression, activation, stimulation, etc.) as well as being useful asan animal model for studying physiological functions of human CTGF.Specifically, it is possible to assess the effect of such apharmaceutical agent on human CTGF by administering the agent to thenon-human transgenic mammal and assaying the levels of human CTGFexpressed in the animal, by using the assay system (sandwich ELISA,etc.) of the present invention.

SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 27 <210> SEQ ID NO 1 <211>LENGTH: 2338 <212> TYPE: DNA <213> ORGANISM: Rat <220> FEATURE: <221>NAME/KEY: 5′UTR <222> LOCATION: (1)..(212) <221> NAME/KEY: CDS <222>LOCATION: (213)..(1256) <221> NAME/KEY: 3′UTR <222> LOCATION:(1257)..(2338) <221> NAME/KEY: polyA_signal <222> LOCATION:(2297)..(2302) <400> SEQUENCE: 1 ctccaagaag actcagccag acccactccagctccgaccc taggagaccg acctcctcca 60 gacggcagca gccccagccc agtggacaaccccaggagcc accacctgga gcgtccggac 120 accaacctcc gccccgagac cgagtccaggctccggccgc gcccctcgtc gcctctgcac 180 cccgctgtgc gtcctcctgc cgcgccccga ccatg ctc gcc tcc gtc gcg ggt 233 Met Leu Ala Ser Val Ala Gly 1 5 ccc gttagc ctc gcc ttg gtg ctc ctc ctc tgc acc cgg cct gcc acc 281 Pro Val SerLeu Ala Leu Val Leu Leu Leu Cys Thr Arg Pro Ala Thr 10 15 20 ggc cag gactgc agc gcg cag tgt cag tgc gca cgt gaa gcg gcg ccg 329 Gly Gln Asp CysSer Ala Gln Cys Gln Cys Ala Arg Glu Ala Ala Pro 25 30 35 cgc tgc ccc gccggc gtg agc ctg gtg ctg gac ggc tgc ggc tgc tgc 377 Arg Cys Pro Ala GlyVal Ser Leu Val Leu Asp Gly Cys Gly Cys Cys 40 45 50 55 cgc gtc tgc gccaag cag ctg gga gaa ctg tgc acg gag cgt gat ccc 425 Arg Val Cys Ala LysGln Leu Gly Glu Leu Cys Thr Glu Arg Asp Pro 60 65 70 tgc gac cca cac aagggt ctc ttc tgc gac ttc ggc tcc ccc gcc aac 473 Cys Asp Pro His Lys GlyLeu Phe Cys Asp Phe Gly Ser Pro Ala Asn 75 80 85 cgc aag att ggc gtg tgccct gcc aaa gat ggt gca ccc tgt gtc ttc 521 Arg Lys Ile Gly Val Cys ProAla Lys Asp Gly Ala Pro Cys Val Phe 90 95 100 ggt ggg tcc gtg tac cgcagc ggc gag tcc ttc caa agc agt tgc aaa 569 Gly Gly Ser Val Tyr Arg SerGly Glu Ser Phe Gln Ser Ser Cys Lys 105 110 115 tac cag tgc act tgc ctggat ggg gcc gtg ggc tgt gtg ccc ctg tgc 617 Tyr Gln Cys Thr Cys Leu AspGly Ala Val Gly Cys Val Pro Leu Cys 120 125 130 135 agc atg gac gtg cgcctg ccc agc cct gac tgc ccc ttc ccg aga agg 665 Ser Met Asp Val Arg LeuPro Ser Pro Asp Cys Pro Phe Pro Arg Arg 140 145 150 gtc aag ctg ccc gggaaa tgc tgt gag gag tgg gtg tgt gat gag ccc 713 Val Lys Leu Pro Gly LysCys Cys Glu Glu Trp Val Cys Asp Glu Pro 155 160 165 aag gac cgc aca gtggtt ggc cct gcc cta gct gcc tac cga ctg gaa 761 Lys Asp Arg Thr Val ValGly Pro Ala Leu Ala Ala Tyr Arg Leu Glu 170 175 180 gac aca ttt ggc cctgac cca act atg atg cga gcc aac tgc ctg gtc 809 Asp Thr Phe Gly Pro AspPro Thr Met Met Arg Ala Asn Cys Leu Val 185 190 195 cag acc aca gag tggagc gcc tgt tct aag acc tgt ggg atg ggc atc 857 Gln Thr Thr Glu Trp SerAla Cys Ser Lys Thr Cys Gly Met Gly Ile 200 205 210 215 tcc acc cgg gttacc aat gac aat acc ttc tgc agg ctg gag aag cag 905 Ser Thr Arg Val ThrAsn Asp Asn Thr Phe Cys Arg Leu Glu Lys Gln 220 225 230 agt cgt ctc tgcatg gtc agg ccc tgt gaa gct gac cta gag gaa aac 953 Ser Arg Leu Cys MetVal Arg Pro Cys Glu Ala Asp Leu Glu Glu Asn 235 240 245 att aag aag ggcaaa aag tgc atc cgg acg cct aaa att gcc aag cct 1001 Ile Lys Lys Gly LysLys Cys Ile Arg Thr Pro Lys Ile Ala Lys Pro 250 255 260 gtc aag ttt gagctt tct ggc tgc acc agt gtg aag acc tac cgg gct 1049 Val Lys Phe Glu LeuSer Gly Cys Thr Ser Val Lys Thr Tyr Arg Ala 265 270 275 aag ttc tgt ggggtg tgc acg gac ggc cgc tgc tgc aca ccg cac aga 1097 Lys Phe Cys Gly ValCys Thr Asp Gly Arg Cys Cys Thr Pro His Arg 280 285 290 295 acc acc acactg ccg gtg gag ttc aag tgc ccc gat ggc gag atc atg 1145 Thr Thr Thr LeuPro Val Glu Phe Lys Cys Pro Asp Gly Glu Ile Met 300 305 310 aaa aag aacatg atg ttc atc aag acc tgt gcc tgc cat tac aac tgt 1193 Lys Lys Asn MetMet Phe Ile Lys Thr Cys Ala Cys His Tyr Asn Cys 315 320 325 ccc ggg gacaat gac atc ttt gag tcc ttg tac tac agg aag atg tat 1241 Pro Gly Asp AsnAsp Ile Phe Glu Ser Leu Tyr Tyr Arg Lys Met Tyr 330 335 340 gga gac atggcg taa agccagggag taagggacac gaactcattt agactataac 1296 Gly Asp Met Ala345 ttgaactgag ttacatctca ttttcttctg taaaaaaaac aaaaagggtt acagtagcac1356 attaatttaa atctgggttc ctaactgctg tgggagaaaa caccccaccg aagtgagaac1416 cgtgtgtcat tgtcatgcaa atagcctgtc aatctcagac actggtttcg agacagttta1476 gacttgacag ttgttcacta gcgcacagtg acagaacgca cactaaggtg agcctcctgg1536 aagagtggag atgccaggag aaagacaggt actagctgag gtcattttaa aagcagcgat1596 atgcctactt tttggagtgt gacaggggag ggacattata gcttgcttgc agacagacct1656 gctctagcaa gagctgggtg tgtgtcctcc actcggtgag gctgaagcca gctattcttt1716 cagtaagaac agcagtttca gcgctgacat tctgattcca gygacactgg tcgggagtca1776 gaaccttgtc tattagactg gacagcttgt ggcaagtgaa tttgccggta acaagccaga1836 tttttatgga tcttgtaaat attgtggata aatatatata tttgtacagt tatctargtt1896 aatttaaaga cgtttgtgcc tattgttctt gttttaagtg cttttggaat ttttaaactg1956 atagcctcaa actccaaaca ccatcgatag gacataaagc ttgtctgtga ttcaaaacaa2016 aggagatact gcagtggaaa ctgtaacctg agtgactgtc tgtcagaaca tatggtacgt2076 agacggtaaa gcaatggatc agaagtcaga tttctagtag gaaatgtaaa atcactgttg2136 gcgaacaaat ggcctttatt aagaaatggc ttgctcaggg taactggtca gatttccacg2196 aggaagtgtt tgctgcttct ttgactatga ctggtttggg aggcagttta tttgttgaga2256 gtgtgaccaa aagttacatg tttgcacctt tctagttgaa aataaagtat atatattttt2316 tatatgaaaa aaaaaaaaaa aa 2338 <210> SEQ ID NO 2 <211> LENGTH: 347<212> TYPE: PRT <213> ORGANISM: Rat <400> SEQUENCE: 2 Met Leu Ala SerVal Ala Gly Pro Val Ser Leu Ala Leu Val Leu Leu 1 5 10 15 Leu Cys ThrArg Pro Ala Thr Gly Gln Asp Cys Ser Ala Gln Cys Gln 20 25 30 Cys Ala ArgGlu Ala Ala Pro Arg Cys Pro Ala Gly Val Ser Leu Val 35 40 45 Leu Asp GlyCys Gly Cys Cys Arg Val Cys Ala Lys Gln Leu Gly Glu 50 55 60 Leu Cys ThrGlu Arg Asp Pro Cys Asp Pro His Lys Gly Leu Phe Cys 65 70 75 80 Asp PheGly Ser Pro Ala Asn Arg Lys Ile Gly Val Cys Pro Ala Lys 85 90 95 Asp GlyAla Pro Cys Val Phe Gly Gly Ser Val Tyr Arg Ser Gly Glu 100 105 110 SerPhe Gln Ser Ser Cys Lys Tyr Gln Cys Thr Cys Leu Asp Gly Ala 115 120 125Val Gly Cys Val Pro Leu Cys Ser Met Asp Val Arg Leu Pro Ser Pro 130 135140 Asp Cys Pro Phe Pro Arg Arg Val Lys Leu Pro Gly Lys Cys Cys Glu 145150 155 160 Glu Trp Val Cys Asp Glu Pro Lys Asp Arg Thr Val Val Gly ProAla 165 170 175 Leu Ala Ala Tyr Arg Leu Glu Asp Thr Phe Gly Pro Asp ProThr Met 180 185 190 Met Arg Ala Asn Cys Leu Val Gln Thr Thr Glu Trp SerAla Cys Ser 195 200 205 Lys Thr Cys Gly Met Gly Ile Ser Thr Arg Val ThrAsn Asp Asn Thr 210 215 220 Phe Cys Arg Leu Glu Lys Gln Ser Arg Leu CysMet Val Arg Pro Cys 225 230 235 240 Glu Ala Asp Leu Glu Glu Asn Ile LysLys Gly Lys Lys Cys Ile Arg 245 250 255 Thr Pro Lys Ile Ala Lys Pro ValLys Phe Glu Leu Ser Gly Cys Thr 260 265 270 Ser Val Lys Thr Tyr Arg AlaLys Phe Cys Gly Val Cys Thr Asp Gly 275 280 285 Arg Cys Cys Thr Pro HisArg Thr Thr Thr Leu Pro Val Glu Phe Lys 290 295 300 Cys Pro Asp Gly GluIle Met Lys Lys Asn Met Met Phe Ile Lys Thr 305 310 315 320 Cys Ala CysHis Tyr Asn Cys Pro Gly Asp Asn Asp Ile Phe Glu Ser 325 330 335 Leu TyrTyr Arg Lys Met Tyr Gly Asp Met Ala 340 345 <210> SEQ ID NO 3 <211>LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence:Artificially synthesized primer sequence <221> NAME/KEY: primer_bind<222> LOCATION: (1)..(20) <400> SEQUENCE: 3 tgcggctgct gccgcgtctg 20<210> SEQ ID NO 4 <211> LENGTH: 21 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Descriptionof Artificial Sequence: Artificially synthesized primer sequence <221>NAME/KEY: primer_bind <222> LOCATION: (1)..(21) <400> SEQUENCE: 4gcacaggtct tgatgaacat c 21 <210> SEQ ID NO 5 <211> LENGTH: 444 <212>TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY:CDS <222> LOCATION: (1)..(444) <221> NAME/KEY: sig_peptide <222>LOCATION: (1)..(57) <221> NAME/KEY: V_region <222> LOCATION: (58)..(363)<400> SEQUENCE: 5 atg gag ttt ggg ctg agc tgg att ttc ctt gct gct atttta aaa ggt 48 Met Glu Phe Gly Leu Ser Trp Ile Phe Leu Ala Ala Ile LeuLys Gly 1 5 10 15 gtc cag tgt gag gtg cag ctg gtg gag tct ggg gga ggcttg gta aag 96 Val Gln Cys Glu Val Gln Leu Val Glu Ser Gly Gly Gly LeuVal Lys 20 25 30 cct ggg ggg tcc ctt aag acc tct cct gtg cag cct ctg gattca act 144 Pro Gly Gly Ser Leu Lys Thr Ser Pro Val Gln Pro Leu Asp SerThr 35 40 45 ttc agt aac gcc tgg atg agc tgg gtc cgc cag gct cca gga aggggc 192 Phe Ser Asn Ala Trp Met Ser Trp Val Arg Gln Ala Pro Gly Arg Gly50 55 60 tgg agt ggg ttg gcc gta tta aaa gca aaa ctg atg gtg gga cac aca240 Trp Ser Gly Leu Ala Val Leu Lys Ala Lys Leu Met Val Gly His Thr 6570 75 80 gac tac gct gca ccc gtg aaa ggc aga ttc acc atc tca aga gat gat288 Asp Tyr Ala Ala Pro Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp 8590 95 tca aaa aac acg ctg tat ctg caa atg aac agc ctg aaa acc gag gac336 Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp 100105 110 aca gcc gtg tat tac tgt acc aca aaa tgg gtg gct acg gac tac ttt384 Thr Ala Val Tyr Tyr Cys Thr Thr Lys Trp Val Ala Thr Asp Tyr Phe 115120 125 gac tac tgg ggc cag gga acc ctg gtc acc gtc tcc tca gcc tcc acc432 Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr 130135 140 aag ggc cca tcg 444 Lys Gly Pro Ser 145 <210> SEQ ID NO 6 <211>LENGTH: 148 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE:6 Met Glu Phe Gly Leu Ser Trp Ile Phe Leu Ala Ala Ile Leu Lys Gly 1 5 1015 Val Gln Cys Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys 20 2530 Pro Gly Gly Ser Leu Lys Thr Ser Pro Val Gln Pro Leu Asp Ser Thr 35 4045 Phe Ser Asn Ala Trp Met Ser Trp Val Arg Gln Ala Pro Gly Arg Gly 50 5560 Trp Ser Gly Leu Ala Val Leu Lys Ala Lys Leu Met Val Gly His Thr 65 7075 80 Asp Tyr Ala Ala Pro Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp 8590 95 Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp100 105 110 Thr Ala Val Tyr Tyr Cys Thr Thr Lys Trp Val Ala Thr Asp TyrPhe 115 120 125 Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser AlaSer Thr 130 135 140 Lys Gly Pro Ser 145 <210> SEQ ID NO 7 <211> LENGTH:447 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE: <221>NAME/KEY: CDS <222> LOCATION: (1)..(447) <221> NAME/KEY: sig_peptide<222> LOCATION: (1)..(57) <221> NAME/KEY: V_region <222> LOCATION:(58)..(357) <400> SEQUENCE: 7 atg gac tgg acc tgg agg atc tct ttc ttggtg gca gca gcc aca gga 48 Met Asp Trp Thr Trp Arg Ile Ser Phe Leu ValAla Ala Ala Thr Gly 1 5 10 15 gcc cac tcc cag gtg cag ctg gtg cag tctggg gct gag gtg aag aag 96 Ala His Ser Gln Val Gln Leu Val Gln Ser GlyAla Glu Val Lys Lys 20 25 30 cct ggg gcc tca gtg aag gtc tcc tgc aag gctttc tgg cta cac ctt 144 Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala PheTrp Leu His Leu 35 40 45 tca ccc ggc tac tat atg cac tgg gtg cga cag gcccct gga caa ggg 192 Ser Pro Gly Tyr Tyr Met His Trp Val Arg Gln Ala ProGly Gln Gly 50 55 60 ctt gag tgg atg gga tgg atc aac cct aac agt agt ggcaca cac tat 240 Leu Glu Trp Met Gly Trp Ile Asn Pro Asn Ser Ser Gly ThrHis Tyr 65 70 75 80 gca cag atg ttt cag ggc agg gtc acc gtg acc agg gacacg tcc atc 288 Ala Gln Met Phe Gln Gly Arg Val Thr Val Thr Arg Asp ThrSer Ile 85 90 95 agc aca gcc tac atg gag ctg agc agg ctg aga tct gac gacacg gcc 336 Ser Thr Ala Tyr Met Glu Leu Ser Arg Leu Arg Ser Asp Asp ThrAla 100 105 110 gtg tat tac tgt gcg aga gag ggg ata gca gca gct gcc atctac ggt 384 Val Tyr Tyr Cys Ala Arg Glu Gly Ile Ala Ala Ala Ala Ile TyrGly 115 120 125 atg gac gtc tgg ggc caa ggg acc acg gtc acc gtc tcc tcagcc tcc 432 Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser AlaSer 130 135 140 acc aag ggc cca tcg 447 Thr Lys Gly Pro Ser 145 <210>SEQ ID NO 8 <211> LENGTH: 149 <212> TYPE: PRT <213> ORGANISM: Homosapiens <400> SEQUENCE: 8 Met Asp Trp Thr Trp Arg Ile Ser Phe Leu ValAla Ala Ala Thr Gly 1 5 10 15 Ala His Ser Gln Val Gln Leu Val Gln SerGly Ala Glu Val Lys Lys 20 25 30 Pro Gly Ala Ser Val Lys Val Ser Cys LysAla Phe Trp Leu His Leu 35 40 45 Ser Pro Gly Tyr Tyr Met His Trp Val ArgGln Ala Pro Gly Gln Gly 50 55 60 Leu Glu Trp Met Gly Trp Ile Asn Pro AsnSer Ser Gly Thr His Tyr 65 70 75 80 Ala Gln Met Phe Gln Gly Arg Val ThrVal Thr Arg Asp Thr Ser Ile 85 90 95 Ser Thr Ala Tyr Met Glu Leu Ser ArgLeu Arg Ser Asp Asp Thr Ala 100 105 110 Val Tyr Tyr Cys Ala Arg Glu GlyIle Ala Ala Ala Ala Ile Tyr Gly 115 120 125 Met Asp Val Trp Gly Gln GlyThr Thr Val Thr Val Ser Ser Ala Ser 130 135 140 Thr Lys Gly Pro Ser 145<210> SEQ ID NO 9 <211> LENGTH: 438 <212> TYPE: DNA <213> ORGANISM: Homosapiens <220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (1)..(438)<221> NAME/KEY: sig_peptide <222> LOCATION: (1)..(57) <221> NAME/KEY:V_region <222> LOCATION: (58)..(350) <400> SEQUENCE: 9 atg gac tgc acctgg agg atc ctc ttc ttg gtg gca gca gct aca ggc 48 Met Asp Cys Thr TrpArg Ile Leu Phe Leu Val Ala Ala Ala Thr Gly 1 5 10 15 acc cac gcc caggtc cag ctg gta cag ttt ggg gct gag gtg aag aag 96 Thr His Ala Gln ValGln Leu Val Gln Phe Gly Ala Glu Val Lys Lys 20 25 30 cct ggg gcc tca gtgaag gtc tcc tgc aag gtt tcc gga tac acc ctc 144 Pro Gly Ala Ser Val LysVal Ser Cys Lys Val Ser Gly Tyr Thr Leu 35 40 45 act gaa tta tcc atg cactgg gtg cga cag gct cct gga aaa ggg ctt 192 Thr Glu Leu Ser Met His TrpVal Arg Gln Ala Pro Gly Lys Gly Leu 50 55 60 gag tgg atg gga agt ttt gatcct gaa gat ggt gaa aca atc tac gca 240 Glu Trp Met Gly Ser Phe Asp ProGlu Asp Gly Glu Thr Ile Tyr Ala 65 70 75 80 cag aag ttc cag ggc aga gtcacc atg acc gag gac aca tct aca gac 288 Gln Lys Phe Gln Gly Arg Val ThrMet Thr Glu Asp Thr Ser Thr Asp 85 90 95 aca gcc tac atg gag ctg agc agcctg aga tct gag gac acg gcc gtg 336 Thr Ala Tyr Met Glu Leu Ser Ser LeuArg Ser Glu Asp Thr Ala Val 100 105 110 tat tac tgt gca acc tct acg gtggta act ccg tgg tac ttt gac tac 384 Tyr Tyr Cys Ala Thr Ser Thr Val ValThr Pro Trp Tyr Phe Asp Tyr 115 120 125 tgg ggc cag gga acc ctg gtc accgtc tcc tca gcc tcc acc aag ggc 432 Trp Gly Gln Gly Thr Leu Val Thr ValSer Ser Ala Ser Thr Lys Gly 130 135 140 cca tcg 438 Pro Ser 145 <210>SEQ ID NO 10 <211> LENGTH: 146 <212> TYPE: PRT <213> ORGANISM: Homosapiens <400> SEQUENCE: 10 Met Asp Cys Thr Trp Arg Ile Leu Phe Leu ValAla Ala Ala Thr Gly 1 5 10 15 Thr His Ala Gln Val Gln Leu Val Gln PheGly Ala Glu Val Lys Lys 20 25 30 Pro Gly Ala Ser Val Lys Val Ser Cys LysVal Ser Gly Tyr Thr Leu 35 40 45 Thr Glu Leu Ser Met His Trp Val Arg GlnAla Pro Gly Lys Gly Leu 50 55 60 Glu Trp Met Gly Ser Phe Asp Pro Glu AspGly Glu Thr Ile Tyr Ala 65 70 75 80 Gln Lys Phe Gln Gly Arg Val Thr MetThr Glu Asp Thr Ser Thr Asp 85 90 95 Thr Ala Tyr Met Glu Leu Ser Ser LeuArg Ser Glu Asp Thr Ala Val 100 105 110 Tyr Tyr Cys Ala Thr Ser Thr ValVal Thr Pro Trp Tyr Phe Asp Tyr 115 120 125 Trp Gly Gln Gly Thr Leu ValThr Val Ser Ser Ala Ser Thr Lys Gly 130 135 140 Pro Ser 145 <210> SEQ IDNO 11 <211> LENGTH: 438 <212> TYPE: DNA <213> ORGANISM: Homo sapiens<220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (1)..(438) <221>NAME/KEY: sig_peptide <222> LOCATION: (1)..(57) <221> NAME/KEY: V_region<222> LOCATION: (58)..(350) <400> SEQUENCE: 11 atg gac tgc acc tgg aggatc ttc ttc ttg gtg gca gca gct aca ggc 48 Met Asp Cys Thr Trp Arg IlePhe Phe Leu Val Ala Ala Ala Thr Gly 1 5 10 15 acc cac gcc cag gtc cagctg gta cag tct ggg gct gag gtg aag aag 96 Thr His Ala Gln Val Gln LeuVal Gln Ser Gly Ala Glu Val Lys Lys 20 25 30 cct ggg gcc tca gtg aag gtctcc tgc aag gtt tcc gga tac acc ctc 144 Pro Gly Ala Ser Val Lys Val SerCys Lys Val Ser Gly Tyr Thr Leu 35 40 45 act gaa tta tcc atg cac tgg gtgcga cag gct cct gga aaa ggg ctt 192 Thr Glu Leu Ser Met His Trp Val ArgGln Ala Pro Gly Lys Gly Leu 50 55 60 gag tgg atg gga agt ttt gat cct gaagat ggt gaa aca atc tac gca 240 Glu Trp Met Gly Ser Phe Asp Pro Glu AspGly Glu Thr Ile Tyr Ala 65 70 75 80 cag aag ttc cag ggc aga gtc acc atgacc gag gac aca tct aca gac 288 Gln Lys Phe Gln Gly Arg Val Thr Met ThrGlu Asp Thr Ser Thr Asp 85 90 95 aca gcc tac atg gag ctg agc agc ctg agatct gag gac acg gcc gtg 336 Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg SerGlu Asp Thr Ala Val 100 105 110 tat tac tgt gca acc tct acg gtg gta actccg tgg tac ttt gac tac 384 Tyr Tyr Cys Ala Thr Ser Thr Val Val Thr ProTrp Tyr Phe Asp Tyr 115 120 125 tgg ggc cag gga acc ctg gtc acc gtc tcctca gcc tcc acc aag ggc 432 Trp Gly Gln Gly Thr Leu Val Thr Val Ser SerAla Ser Thr Lys Gly 130 135 140 cca tcg 438 Pro Ser 145 <210> SEQ ID NO12 <211> LENGTH: 146 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <400>SEQUENCE: 12 Met Asp Cys Thr Trp Arg Ile Phe Phe Leu Val Ala Ala Ala ThrGly 1 5 10 15 Thr His Ala Gln Val Gln Leu Val Gln Ser Gly Ala Glu ValLys Lys 20 25 30 Pro Gly Ala Ser Val Lys Val Ser Cys Lys Val Ser Gly TyrThr Leu 35 40 45 Thr Glu Leu Ser Met His Trp Val Arg Gln Ala Pro Gly LysGly Leu 50 55 60 Glu Trp Met Gly Ser Phe Asp Pro Glu Asp Gly Glu Thr IleTyr Ala 65 70 75 80 Gln Lys Phe Gln Gly Arg Val Thr Met Thr Glu Asp ThrSer Thr Asp 85 90 95 Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu AspThr Ala Val 100 105 110 Tyr Tyr Cys Ala Thr Ser Thr Val Val Thr Pro TrpTyr Phe Asp Tyr 115 120 125 Trp Gly Gln Gly Thr Leu Val Thr Val Ser SerAla Ser Thr Lys Gly 130 135 140 Pro Ser 145 <210> SEQ ID NO 13 <211>LENGTH: 450 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE:<221> NAME/KEY: CDS <222> LOCATION: (1)..(450) <221> NAME/KEY:sig_peptide <222> LOCATION: (1)..(58) <221> NAME/KEY: V_region <222>LOCATION: (59)..(353) <400> SEQUENCE: 13 atg aaa cac ctg tgg ttc ttc cttcct gct ggt ggc agc tcc cag atg 48 Met Lys His Leu Trp Phe Phe Leu ProAla Gly Gly Ser Ser Gln Met 1 5 10 15 ggt cct gtc cca ggt gca gct gcagga gtc ggg ccc agg act ggt gaa 96 Gly Pro Val Pro Gly Ala Ala Ala GlyVal Gly Pro Arg Thr Gly Glu 20 25 30 gcc ttc aca gac cct gtc ctc acc tgcact gtc tct ggt ggc tcc atc 144 Ala Phe Thr Asp Pro Val Leu Thr Cys ThrVal Ser Gly Gly Ser Ile 35 40 45 agc agt ggt ggt tac tac tgg agc tgg atccgc cag cac cca ggg aag 192 Ser Ser Gly Gly Tyr Tyr Trp Ser Trp Ile ArgGln His Pro Gly Lys 50 55 60 ggc ctg gag tgg att ggg tac atc tat tac agtggg agc acc tac tac 240 Gly Leu Glu Trp Ile Gly Tyr Ile Tyr Tyr Ser GlySer Thr Tyr Tyr 65 70 75 80 aac ccg tcc ctc aag agt cga gtt acc ata tcagta gac acg tct aag 288 Asn Pro Ser Leu Lys Ser Arg Val Thr Ile Ser ValAsp Thr Ser Lys 85 90 95 aac cag ttc tcc ctg aag ctg agc tct gtg act gccgcg gac acg gcc 336 Asn Gln Phe Ser Leu Lys Leu Ser Ser Val Thr Ala AlaAsp Thr Ala 100 105 110 gtg tat tac tgt gcg agc tat tac tat gat agt ggtggt tat tac gac 384 Val Tyr Tyr Cys Ala Ser Tyr Tyr Tyr Asp Ser Gly GlyTyr Tyr Asp 115 120 125 tac ttt gac tac tgg ggc cag gga acc ctg gtc accgtc tcc tca gcc 432 Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr ValSer Ser Ala 130 135 140 tcc acc aag ggc cca tcg 450 Ser Thr Lys Gly ProSer 145 150 <210> SEQ ID NO 14 <211> LENGTH: 150 <212> TYPE: PRT <213>ORGANISM: Homo sapiens <400> SEQUENCE: 14 Met Lys His Leu Trp Phe PheLeu Pro Ala Gly Gly Ser Ser Gln Met 1 5 10 15 Gly Pro Val Pro Gly AlaAla Ala Gly Val Gly Pro Arg Thr Gly Glu 20 25 30 Ala Phe Thr Asp Pro ValLeu Thr Cys Thr Val Ser Gly Gly Ser Ile 35 40 45 Ser Ser Gly Gly Tyr TyrTrp Ser Trp Ile Arg Gln His Pro Gly Lys 50 55 60 Gly Leu Glu Trp Ile GlyTyr Ile Tyr Tyr Ser Gly Ser Thr Tyr Tyr 65 70 75 80 Asn Pro Ser Leu LysSer Arg Val Thr Ile Ser Val Asp Thr Ser Lys 85 90 95 Asn Gln Phe Ser LeuLys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala 100 105 110 Val Tyr Tyr CysAla Ser Tyr Tyr Tyr Asp Ser Gly Gly Tyr Tyr Asp 115 120 125 Tyr Phe AspTyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala 130 135 140 Ser ThrLys Gly Pro Ser 145 150 <210> SEQ ID NO 15 <211> LENGTH: 423 <212> TYPE:DNA <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: CDS<222> LOCATION: (1)..(423) <221> NAME/KEY: sig_peptide <222> LOCATION:(1)..(60) <221> NAME/KEY: V_region <222> LOCATION: (61)..(365) <400>SEQUENCE: 15 atg gtg ttg cag acc cag gtc ttc att tct ctg ttg ctc tgg atctct 48 Met Val Leu Gln Thr Gln Val Phe Ile Ser Leu Leu Leu Trp Ile Ser 15 10 15 ggt gcc tac ggg gac atc gtg atg acc cag tct cca gac tcc ctg gct96 Gly Ala Tyr Gly Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala 20 2530 gtg tct ctg ggc gag agg gcc acc atc aac tgc aag tcc agc cag act 144Val Ser Leu Gly Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Thr 35 40 45gtt tta tac agc tcc aac aat aag aac tac tta gct tgg tac cag cag 192 ValLeu Tyr Ser Ser Asn Asn Lys Asn Tyr Leu Ala Trp Tyr Gln Gln 50 55 60 aaacca gga cag cct cct aag ctg ctc att tac tgg gca tct acc cgg 240 Lys ProGly Gln Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg 65 70 75 80 gaatcc ggg gtc cct gac cga ttc agt ggc agc ggg tct ggg aca gat 288 Glu SerGly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp 85 90 95 ttc actctc acc atc agc agc ctg cag gct gac gat gtg gca gtt tat 336 Phe Thr LeuThr Ile Ser Ser Leu Gln Ala Asp Asp Val Ala Val Tyr 100 105 110 tac tgtcag caa tat tat agt act cct ccg tgg acg ttc ggc caa ggg 384 Tyr Cys GlnGln Tyr Tyr Ser Thr Pro Pro Trp Thr Phe Gly Gln Gly 115 120 125 acc aaggtg gaa atc aaa cga act gtg gct gca cca tct 423 Thr Lys Val Glu Ile LysArg Thr Val Ala Ala Pro Ser 130 135 140 <210> SEQ ID NO 16 <211> LENGTH:141 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 16 MetVal Leu Gln Thr Gln Val Phe Ile Ser Leu Leu Leu Trp Ile Ser 1 5 10 15Gly Ala Tyr Gly Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala 20 25 30Val Ser Leu Gly Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Thr 35 40 45Val Leu Tyr Ser Ser Asn Asn Lys Asn Tyr Leu Ala Trp Tyr Gln Gln 50 55 60Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg 65 70 7580 Glu Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp 85 9095 Phe Thr Leu Thr Ile Ser Ser Leu Gln Ala Asp Asp Val Ala Val Tyr 100105 110 Tyr Cys Gln Gln Tyr Tyr Ser Thr Pro Pro Trp Thr Phe Gly Gln Gly115 120 125 Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala Pro Ser 130 135140 <210> SEQ ID NO 17 <211> LENGTH: 420 <212> TYPE: DNA <213> ORGANISM:Homo sapiens <220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION:(1)..(420) <221> NAME/KEY: sig_peptide <222> LOCATION: (1)..(60) <221>NAME/KEY: V_region <222> LOCATION: (61)..(364) <400> SEQUENCE: 17 atgaag gat ctg ctc agc ttc ctg ggg ctg cta atg ctc tgg ata cct 48 Met LysAsp Leu Leu Ser Phe Leu Gly Leu Leu Met Leu Trp Ile Pro 1 5 10 15 ggatcc agt gca gat att gtc atg acc cag acg cca ctc ttc tgt ccg 96 Gly SerSer Ala Asp Ile Val Met Thr Gln Thr Pro Leu Phe Cys Pro 20 25 30 tca cccctg gac agc cga gcc tcc atc tcc tgc aag tct ggt ctg agc 144 Ser Pro LeuAsp Ser Arg Ala Ser Ile Ser Cys Lys Ser Gly Leu Ser 35 40 45 ctc ctg cacagt gat gga aag acc tat ttg cat tgg tac ctg cag aag 192 Leu Leu His SerAsp Gly Lys Thr Tyr Leu His Trp Tyr Leu Gln Lys 50 55 60 cca ggc cag cctcca cag ctc ctg atc tat gag agt ttc caa ccg gtt 240 Pro Gly Gln Pro ProGln Leu Leu Ile Tyr Glu Ser Phe Gln Pro Val 65 70 75 80 ctc ctg gag tgccag ata ggc tca gtg gca gcg ggt cag gac aga ttt 288 Leu Leu Glu Cys GlnIle Gly Ser Val Ala Ala Gly Gln Asp Arg Phe 85 90 95 cac act gaa aat cagccg ggt gga agg ctg agg aat gtt ggg gtt tat 336 His Thr Glu Asn Gln ProGly Gly Arg Leu Arg Asn Val Gly Val Tyr 100 105 110 tac tgc atg caa agttta cag ctt ccg ctc act ttc ggc gga ggg acc 384 Tyr Cys Met Gln Ser LeuGln Leu Pro Leu Thr Phe Gly Gly Gly Thr 115 120 125 aag gtg gag atc aaacga act gtg gct gca cca tct 420 Lys Val Glu Ile Lys Arg Thr Val Ala AlaPro Ser 130 135 140 <210> SEQ ID NO 18 <211> LENGTH: 140 <212> TYPE: PRT<213> ORGANISM: Homo sapiens <400> SEQUENCE: 18 Met Lys Asp Leu Leu SerPhe Leu Gly Leu Leu Met Leu Trp Ile Pro 1 5 10 15 Gly Ser Ser Ala AspIle Val Met Thr Gln Thr Pro Leu Phe Cys Pro 20 25 30 Ser Pro Leu Asp SerArg Ala Ser Ile Ser Cys Lys Ser Gly Leu Ser 35 40 45 Leu Leu His Ser AspGly Lys Thr Tyr Leu His Trp Tyr Leu Gln Lys 50 55 60 Pro Gly Gln Pro ProGln Leu Leu Ile Tyr Glu Ser Phe Gln Pro Val 65 70 75 80 Leu Leu Glu CysGln Ile Gly Ser Val Ala Ala Gly Gln Asp Arg Phe 85 90 95 His Thr Glu AsnGln Pro Gly Gly Arg Leu Arg Asn Val Gly Val Tyr 100 105 110 Tyr Cys MetGln Ser Leu Gln Leu Pro Leu Thr Phe Gly Gly Gly Thr 115 120 125 Lys ValGlu Ile Lys Arg Thr Val Ala Ala Pro Ser 130 135 140 <210> SEQ ID NO 19<211> LENGTH: 405 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220>FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (1)..(405) <221> NAME/KEY:sig_peptide <222> LOCATION: (1)..(66) <221> NAME/KEY: V_region <222>LOCATION: (67)..(353) <400> SEQUENCE: 19 atg gac atg agg gtc cct gct cagctc ctg ggg ctc ctg ctg ctc tgg 48 Met Asp Met Arg Val Pro Ala Gln LeuLeu Gly Leu Leu Leu Leu Trp 1 5 10 15 ctc tca ggt gcc aga tgt gac atccag atg acc cag tct cca tcc ttc 96 Leu Ser Gly Ala Arg Cys Asp Ile GlnMet Thr Gln Ser Pro Ser Phe 20 25 30 cct gtc tgc atc tgt agg aga cag agtcac cat cac ttg cca ggc gag 144 Pro Val Cys Ile Cys Arg Arg Gln Ser HisHis His Leu Pro Gly Glu 35 40 45 tca gga cat tca cca cta ttt aaa ttg gtatca gca gaa acc agg gaa 192 Ser Gly His Ser Pro Leu Phe Lys Leu Val SerAla Glu Thr Arg Glu 50 55 60 agc cct aag ctc ctg atc tac gat gca tcc aatttg gaa aca ggg tcc 240 Ser Pro Lys Leu Leu Ile Tyr Asp Ala Ser Asn LeuGlu Thr Gly Ser 65 70 75 80 cat cac ggt tca gtg gaa gtg gat ctg gga cagatt tta ctt tca cca 288 His His Gly Ser Val Glu Val Asp Leu Gly Gln IleLeu Leu Ser Pro 85 90 95 tca gca gcc tgc agc tct gaa gat att gca aca tattac tgt caa cag 336 Ser Ala Ala Cys Ser Ser Glu Asp Ile Ala Thr Tyr TyrCys Gln Gln 100 105 110 tat aat aat ctc atc acc ttc ggc caa ggg aca cgactg gag att aaa 384 Tyr Asn Asn Leu Ile Thr Phe Gly Gln Gly Thr Arg LeuGlu Ile Lys 115 120 125 cga act gtg gct gca cca tct 405 Arg Thr Val AlaAla Pro Ser 130 135 <210> SEQ ID NO 20 <211> LENGTH: 135 <212> TYPE: PRT<213> ORGANISM: Homo sapiens <400> SEQUENCE: 20 Met Asp Met Arg Val ProAla Gln Leu Leu Gly Leu Leu Leu Leu Trp 1 5 10 15 Leu Ser Gly Ala ArgCys Asp Ile Gln Met Thr Gln Ser Pro Ser Phe 20 25 30 Pro Val Cys Ile CysArg Arg Gln Ser His His His Leu Pro Gly Glu 35 40 45 Ser Gly His Ser ProLeu Phe Lys Leu Val Ser Ala Glu Thr Arg Glu 50 55 60 Ser Pro Lys Leu LeuIle Tyr Asp Ala Ser Asn Leu Glu Thr Gly Ser 65 70 75 80 His His Gly SerVal Glu Val Asp Leu Gly Gln Ile Leu Leu Ser Pro 85 90 95 Ser Ala Ala CysSer Ser Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln 100 105 110 Tyr Asn AsnLeu Ile Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys 115 120 125 Arg ThrVal Ala Ala Pro Ser 130 135 <210> SEQ ID NO 21 <211> LENGTH: 387 <212>TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY:CDS <222> LOCATION: (1)..(387) <221> NAME/KEY: sig_peptide <222>LOCATION: (1)..(47) <223> OTHER INFORMATION: Initiation codon and aportion of a signal sequence are lacked. <221> NAME/KEY: V_region <222>LOCATION: (48)..(335) <400> SEQUENCE: 21 gat agg gtc cta ggg gtc ctg atggtt ggg ttt tcg gtg ccg gat gag 48 Asp Arg Val Leu Gly Val Leu Met ValGly Phe Ser Val Pro Asp Glu 1 5 10 15 aac atc cag atg acc cag tat ccatct ccc tgt ctg cat acc tgt agg 96 Asn Ile Gln Met Thr Gln Tyr Pro SerPro Cys Leu His Thr Cys Arg 20 25 30 aga cag agt cac cat cac ttg cca gagcga gct cag gac att cac cac 144 Arg Gln Ser His His His Leu Pro Glu ArgAla Gln Asp Ile His His 35 40 45 tat cta aat tgg tat cag cag aaa cca gggaaa gcc cta agc tct gat 192 Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly LysAla Leu Ser Ser Asp 50 55 60 cta cga tgc atc caa ttt gga aac agg gtc ccatca cgg ttc agt gga 240 Leu Arg Cys Ile Gln Phe Gly Asn Arg Val Pro SerArg Phe Ser Gly 65 70 75 80 agt gga tct ggg aca gat tct act tca cca tcagca gcc tgc agc tct 288 Ser Gly Ser Gly Thr Asp Ser Thr Ser Pro Ser AlaAla Cys Ser Ser 85 90 95 gaa gat att gca aca tat tac tgt caa cag tat aataat ctc atc acc 336 Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn AsnLeu Ile Thr 100 105 110 ttc ggc caa ggg aca cga ctg gag att aaa cga actgtg gct gca cca 384 Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys Arg Thr ValAla Ala Pro 115 120 125 tct 387 Ser <210> SEQ ID NO 22 <211> LENGTH: 129<212> TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 22 Asp ArgVal Leu Gly Val Leu Met Val Gly Phe Ser Val Pro Asp Glu 1 5 10 15 AsnIle Gln Met Thr Gln Tyr Pro Ser Pro Cys Leu His Thr Cys Arg 20 25 30 ArgGln Ser His His His Leu Pro Glu Arg Ala Gln Asp Ile His His 35 40 45 TyrLeu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Leu Ser Ser Asp 50 55 60 LeuArg Cys Ile Gln Phe Gly Asn Arg Val Pro Ser Arg Phe Ser Gly 65 70 75 80Ser Gly Ser Gly Thr Asp Ser Thr Ser Pro Ser Ala Ala Cys Ser Ser 85 90 95Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Asn Leu Ile Thr 100 105110 Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys Arg Thr Val Ala Ala Pro 115120 125 Ser <210> SEQ ID NO 23 <211> LENGTH: 411 <212> TYPE: DNA <213>ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: CDS <222>LOCATION: (1)..(411) <221> NAME/KEY: sig_peptide <222> LOCATION:(1)..(66) <221> NAME/KEY: V_region <222> LOCATION: (67)..(356) <400>SEQUENCE: 23 atg gac atg agg gtc cct gct cag ctc ctg ggg ctc ctg ctg ctctgg 48 Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp 15 10 15 ctc tca ggt gcc aga tgt gac atc cag atg acc cag tct cca tcc tcc96 Leu Ser Gly Ala Arg Cys Asp Ile Gln Met Thr Gln Ser Pro Ser Ser 20 2530 ctg tct gca tct gta gga gac aga gtc acc atc act tgc cgg gca agt 144Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser 35 40 45cag agc att agc agc tat tta aat tgg tat cag cag aaa cca ggg aaa 192 GlnSer Ile Ser Ser Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys 50 55 60 gcccct aag ctc ctg att tat gct gca tcc agt ttg caa agt ggg tcc 240 Ala ProLys Leu Leu Ile Tyr Ala Ala Ser Ser Leu Gln Ser Gly Ser 65 70 75 80 catcaa ggt tca gtg gca gtg gat tat gcg aca gat ttc cat ttc tca 288 His GlnGly Ser Val Ala Val Asp Tyr Ala Thr Asp Phe His Phe Ser 85 90 95 cca tcagca gtt tgc cac ctg acg att ttg caa ctt act act gtc cac 336 Pro Ser AlaVal Cys His Leu Thr Ile Leu Gln Leu Thr Thr Val His 100 105 110 aga gttaca gta tcc cat tca ctt tcg gcc ctg ggg acc aaa gtg gat 384 Arg Val ThrVal Ser His Ser Leu Ser Ala Leu Gly Thr Lys Val Asp 115 120 125 agc aaacga act gtg gct gca cca tct 411 Ser Lys Arg Thr Val Ala Ala Pro Ser 130135 <210> SEQ ID NO 24 <211> LENGTH: 137 <212> TYPE: PRT <213> ORGANISM:Homo sapiens <400> SEQUENCE: 24 Met Asp Met Arg Val Pro Ala Gln Leu LeuGly Leu Leu Leu Leu Trp 1 5 10 15 Leu Ser Gly Ala Arg Cys Asp Ile GlnMet Thr Gln Ser Pro Ser Ser 20 25 30 Leu Ser Ala Ser Val Gly Asp Arg ValThr Ile Thr Cys Arg Ala Ser 35 40 45 Gln Ser Ile Ser Ser Tyr Leu Asn TrpTyr Gln Gln Lys Pro Gly Lys 50 55 60 Ala Pro Lys Leu Leu Ile Tyr Ala AlaSer Ser Leu Gln Ser Gly Ser 65 70 75 80 His Gln Gly Ser Val Ala Val AspTyr Ala Thr Asp Phe His Phe Ser 85 90 95 Pro Ser Ala Val Cys His Leu ThrIle Leu Gln Leu Thr Thr Val His 100 105 110 Arg Val Thr Val Ser His SerLeu Ser Ala Leu Gly Thr Lys Val Asp 115 120 125 Ser Lys Arg Thr Val AlaAla Pro Ser 130 135 <210> SEQ ID NO 25 <211> LENGTH: 27 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence: Artificiallysynthesized adaptor sequence <221> NAME/KEY: misc_difference <222>LOCATION: (1)..(27) <400> SEQUENCE: 25 ccatcctaat acgactcact atagggc 27<210> SEQ ID NO 26 <211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Descriptionof Artificial Sequence: Artificially synthesized primer sequence <221>NAME/KEY: primer_bind <222> LOCATION: (1)..(25) <400> SEQUENCE: 26ccagggccgc tgtgctctcg gaggt 25 <210> SEQ ID NO 27 <211> LENGTH: 23 <212>TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence: Artificiallysynthesized primer sequence <221> NAME/KEY: primer_bind <222> LOCATION:(1)..(23) <400> SEQUENCE: 27 gggggtcagg ctggaactga gga 23

What is claimed is:
 1. A non-human monoclonal antibody or a portionthereof selected from the group consisting of F(ab′)2, Fab, Fab′, Fv,sFv, dsFv and dAb, which (a) binds to human, mouse and rat connectivetissue growth factors (CTGFs) and (b) has the IgG isotype.
 2. Thenon-human monoclonal antibody or a portion thereof according to claim 1,wherein said antibody inhibits the binding of human CTGF to humankidney-derived fibroblast cell line 293-T (ATCC CRL 1573).
 3. Thenon-human monoclonal antibody or a portion thereof according to claim 1,wherein said antibody is a mouse, rat or hamster antibody.
 4. Thenon-human monoclonal antibody or a portion thereof according to claim 3,wherein said antibody inhibits the binding of human CTGF to humankidney-derived fibroblast cell line 293-T (ATCC CRL 1573).
 5. Anon-human monoclonal antibody which is produced by a hybridomaidentified by international deposit accession numbers selected from thegroup consisting of FERM BP-6208 and FERM BP-6209.
 6. A cell producingthe non-human monoclonal antibody according to claim
 1. 7. The cellaccording to claim 6, wherein said cell is a hybridoma obtained byfusing a mammalian myeloma cell with a mammalian B cell that producesthe non-human monoclonal antibody.
 8. A cell identified by internationaldeposit accession numbers selected from the group consisting of FERMBP-6208 and FERM BP-6209.
 9. An antibody-immobilized insoluble carriercomprising the non-human monoclonal antibody according to claim 1 orclaim
 5. 10. The non-human antibody-immobilized insoluble carrieraccording to claim 9, wherein said insoluble carrier is selected fromthe group consisting of plates, test tubes, tubes, beads, balls, filtersand membranes.
 11. The non-human antibody-immobilized insoluble carrieraccording to claim 9, wherein said insoluble carrier is a filter ormembrane, for affinity column chromatography.
 12. A labeled antibodycomprising the non-human monoclonal antibody or a portion thereofaccording to claim 1 or the non-human monoclonal antibody according toclaim 5 that is labeled with a labeling agent that provides a detectablesignal.
 13. The labeled non-human antibody according to claim 12,wherein said labeling agent is an enzyme, fluorescent substance,chemiluminescent substance, biotin, avidin, or radioisotope.
 14. A kitfor detecting or assaying mammalian CTGF, comprising the non-humanmonoclonal antibody or a portion thereof according to claim 1 or thenon-human monoclonal antibody according to claim
 5. 15. A kit fordetecting or assaying mammalian CTGF comprising an antibody-immobilizedinsoluble carrier which comprises the non-human monoclonal antibodyaccording to claim 1 or claim
 5. 16. A kit for detecting or assayingmammalian CTGF comprising a labeled antibody which comprises thenon-human monoclonal antibody or a portion thereof according to claim 1or the non-human monoclonal antibody according to claim 5 that islabeled with a labeling agent that provides a detectable signal.
 17. Akit for purifying mammalian CTGF, comprising an antibody-immobilizedinsoluble carrier which comprises the non-human monoclonal antibodyaccording to claim 1 or claim 5.